Modification of recombinant adenovirus with immunogenic plasmodium circumsporozoite protein epitopes

ABSTRACT

The present disclosure relates to adenovirus protein modifications to augment immune response to a transgene of a recombinant adenovirus and to circumvent pre-existing anti-adenovirus immunity. Some embodiments are directed to a recombinant adenovirus derived from a recombinant adenovirus plasmid vector, wherein the recombinant adenovirus plasmid vector comprises a nucleotide sequence encoding a  Plasmodium  circumsporozoite protein, or antigenic portion thereof, operably linked to a heterologous promoter and a modified capsid or core protein, wherein an immunogenic epitope of  Plasmodium  circumsporozoite is inserted into or replaces at least part of a capsid or core protein. Other embodiments are directed to a pharmaceutical composition or a malaria vaccine composition comprising a recombinant adenovirus according to the above embodiments. Further embodiments include a method of treating, preventing, or diagnosing malaria, comprising administering a therapeutic amount of the pharmaceutical composition or malaria vaccine composition in accordance with the above embodiment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US10/045,952, filed Aug. 18, 2010, which claims priority to and is acontinuation-in-part of International Application No. PCT/US09/054,212,filed on Aug. 18, 2009, both of which are incorporated by reference intheir entirety, as if fully set forth herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.1R01AI081510-01A1 awarded by the National Institute of Allergy andInfectious Diseases (NIAID), an institute that is part of the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to the field of medicine and biotechnology. Moreparticularly, the invention relates to the use of capsid-modifiedadenoviral vectors to induce a potent immune response to a malariaparasite antigen such as Plasmodium circumsporozoite protein, which aresuitable for vaccines against malaria.

BACKGROUND

Malaria is a severe disease that ranks among the most prevalentinfections in tropical areas throughout the world. Approximately 300-500million people become infected yearly, with relatively high rates ofmorbidity and mortality. Severe morbidity and mortality occurparticularly in young children and in adults migrating to a malariaendemic area without having undergone prior malaria exposure. The WorldHealth Organization (WHO) estimates that 2-3 million children die ofmalaria in Africa alone, every year. The widespread occurrence and theincreasing incidence of malaria in many countries, caused bydrug-resistant parasites (Plasmodium falciparum, recently alsoPlasmodium vivax) and insecticide-resistant vectors (Anophelesmosquitoes), underscore the need for developing new methods for thecontrol of this disease (Nussenzweig and Long 1994).

Malaria parasites have a complicated life cycle consisting ofpre-erythrocytic, erythrocytic and sexual parasitic forms, representinga potential target for the development of a malaria vaccine. Thepre-erythrocytic and erythrocytic forms are found in the host, while thesexual forms occur in the vector. Immunization with live-attenuatedirradiated sporozoites (IrSp) has been shown to induce sterileprotection (i.e., complete resistance against parasite challenge) inmice (Nussenzweig et al. 1967), non-human primates (Gwadz et al. 1979)and human (Clyde et al. 1973, Edelman et al. 1993). Protection conferredby IrSp is mediated by sporozoite neutralization by both humoral (Bcell) and cellular (T cell) immune responses (Tsuji et al. 2001).Although an IrSp vaccination is an attractive solution, the only way toobtain sporozoites is by dissecting mosquito salivary glands, and thereis currently no known technology to grow large numbers of sporozoites invitro. Therefore, an alternate vaccine vector that can elicit an equallystrong protective immunity against malaria is needed.

One promising target for such a vaccine vector is the circumsporozoite(CS) protein, which is expressed on the surface of the sporozoite.Effective neutralizing antibodies are directed against theimmunodominant, species specific, repeat domains of the circumsporozoite(CS) protein. In Plasmodium falciparum (human malaria parasite), therepeats (NANP)_(n) are conserved among isolates from all areas of theworld. This central repeat contains multiple repeat of B cell epitopes,and, therefore, the CS protein can induce a strong humoral immuneresponse by triggering B cells (Tsuji et al. 2001). At the C-terminalregion of the CS protein, there are several T cell epitopes, which caninduce a significant cellular immune response (Tsuji et al. 2001). Thehumoral (antibody) response can eliminate parasites by interacting andneutralizing the infectivity of sporozoites (extra-cellular parasite)prior to entering hepatocyte, whereas the cellular (T cell) response canattack EEF (an intra-cellular parasite) by secreting interferon-gamma.These immune responses prevent the EEFs from maturing and dividingrapidly to form thousands of merozoites that reenter the blood andinfect erythrocytes causing the disease we recognize as malaria.

One CS-based malarial vaccine that is currently undergoing human trialsis GlaxoSmithKline's RTS, S, fusion protein of the Hepatitis B surfaceantigen and a portion of Plasmodium falciparum circumsporozoite protein(PfCSP) in a form of virus-like particle (International PatentApplication No. PCT/EP1992/002591 to SmithKline Beecham BiologicalsS.A., filed Nov. 11, 1992), has been shown to decrease malaria infectionin clinical trials (Alonso et al. 2004, Alonso et al. 2005, Bejon et al.2008). RTS, S induces an anti-PfCSP humoral immune response, but arelatively weak PfCSP-specific cellular (CD8+) response (Kester et al.2008), which might be the reason for the relatively weak protection byRTS, S. In contrast, adenovirus-based malaria vaccines can induce aprotective cellular immune response (International Patent ApplicationNo. PCT/EP2003/051019, filed Dec. 16, 2003, Rodrigues et al. 1997).However, there are currently two obstacles that limit the use of anadenovirus-based platform as a malaria vaccine: (1) lack of a capabilityof inducing a potent humoral response against a transgene product, and(2) pre-existing immunity to adenovirus, especially adenovirus serotype5, which hampers the immunogenicity of adenovirus-based vaccine.

One approach that has recently been taken in an attempt to augmentadenovirus-induced humoral response is to insert a B cell antigenicepitope (e.g., a bacterial or viral epitope) in adenovirus capsidproteins such as Hexon, Fiber, Penton and pIX (Worgall et al. 2005,McConnell et al. 2006, Krause et al. 2006, Worgall et al. 2007).

In addition, to circumvent pre-existing immunity to adenovirus serotype5 (Ad5), other adenovirus serotypes with lower seroprevalence, such asadenovirus serotype 11, 35, 26, 48, 49 and 50, have been evaluated as avaccine platform and shown to induce immune response to a transgene inspite of the presence of anti-Ad5 immunity (International PatentApplication No. PCT/EP2005/055183 to Crucell Holland B.V., filed Oct.12, 2005, Abbink et al. 2007). Substitution of Ad5 Hexon, which is thetarget capsid protein of neutralizing antibody, with that of otherserotypes has also been constructed in order to escape pre-existinganti-Ad5 immunity (Wu et al. 2002, Roberts et al. 2006).

There is, however, no improved adenoviral vector reported to haveovercome the two obstacles at the same time in applying an adenoviralvector to a malaria vaccine mentioned above. Given that seroprevalenceto Ad5 is high in malaria endemic areas (Ophorst et al. 2006.), there isa need for an adenovirus-based malaria vaccine that induces bothprotective humoral and cellular immune responses even in the presence ofpre-exiting immunity to adenovirus.

SUMMARY

The present disclosure relates to various adenovirus proteinmodifications to augment immune response to a transgene of a recombinantadenoviral vaccine and to circumvent pre-existing anti-adenovirusimmunity.

More specifically, one embodiment is directed to a recombinantadenovirus derived from a recombinant adenovirus plasmid vector, whereinthe recombinant adenovirus plasmid vector comprises a nucleotidesequence encoding (i) a Plasmodium circumsporozoite protein, orantigenic portion thereof, operably linked to a heterologous promoter:and (ii) a modified capsid or core protein, wherein an immunogenicepitope of Plasmodium circumsporozoite has been inserted into orreplaces at least part of a capsid or core protein.

In some embodiments, the Plasmodium circumsporozoite protein furthercomprises a Plasmodium falciparum or Plasmodium yoelii circumsporozoiteprotein. The circumsporozoite protein may further comprise acodon-optimized Plasmodium falciparum or Plasmodium yoeliicircumsporozoite protein, and in some aspects, may be encoded by thenucleotide sequence of SEQ ID NO:2 or SEQ ID NO:1, respectively.

In other embodiments, the immunogenic epitope further comprises a B cellepitope of Plasmodium circumsporozoite protein. The B cell epitope maybe incorporated in a modified capsid protein, and in some aspects, thecapsid protein may comprise a Hexon hypervariable region (HVR). The HVRmay further comprise HVR1 or HVR5, wherein a portion of HVR1 or HVR5 isreplaced with the B cell epitope. In other aspects, the capsid proteinmay further comprise a capsid Fiber protein wherein the B cell epitopeis inserted into the Fiber protein. In some aspects, the B cell epitopeis a Plasmodium falciparum circumsporozoite protein B cell epitope,wherein the B cell epitope is a repeat sequence, for example, (NANP)_(n)(SEQ ID NO:60), wherein the repeat sequence may be (NANP)₄, (NANP)₆,(NANP)₈, (NANP)₁₀, (NANP)₁₂, (NANP)₁₄, (NANP)₁₆, (NANP)₁₈, (NANP)₂₀,(NANP)₂₂ or (NANP)₂₈. In other aspects, the B cell epitope is aPlasmodium yoelii circumsporozoite protein B cell epitope, wherein the Bcell epitope is a repeat sequence, for example, (QGPGAP)_(n) (SEQ IDNO:59), wherein the repeat sequence may be (QGPGAP)₃, (QGPGAP)₄,(QGPGAP)₅, (QGPGAP)₆, (QGPGAP)₇, (QGPGAP)₈, (QGPGAP)₉, (QGPGAP)₁₁, or(QGPGAP)₁₂.

In yet other embodiments, the immunogenic epitope further comprises aCD4+ or CD8+ T cell epitope of Plasmodium circumsporozoite protein. TheCD4+ or CD8+ T cell epitope may be incorporated in a modified capsid orcore protein. In some aspects, the capsid protein may comprise a Hexonhypervariable region (HVR). The HVR may further comprise HVR1 wherein aportion of HVR1 is replaced with the CD4+ or CD8+ T cell epitope. Inother aspects, the core protein further comprises a pVII protein and aCD4+ T cell epitope is inserted into the pVII protein. In some aspectsthe CD4+ T cell epitope is a Plasmodium falciparum circumsporozoite CD4+T cell epitope, wherein the CD4+ T cell epitope is EYLNKIQNSLSTEWSPCSVT(SEQ ID NO:62). In other aspects the CD4+ T cell epitope is a Plasmodiumyoelii circumsporozoite CD4+ T cell epitope, wherein the CD4+ T cellepitope is YNRNIVNRLLGDALNGKPEEK (SEQ ID NO:61)

Other embodiments are directed to a pharmaceutical composition ormalaria vaccine composition comprising a recombinant adenovirusaccording to the above embodiments. Further embodiments include a methodof treating, preventing, or diagnosing malaria, comprising administeringa therapeutic amount of the pharmaceutical composition or malariavaccine composition in accordance with the above embodiments.

In another embodiment, a method for treatment comprising administering aprime-boost vaccination, wherein a subject is given a series ofincreasing dosages or same dosages at a given time interval. The timeinterval may be any length sufficient to generate a humoral and/orcellular immune response. For example, as described below, the intervalmay be, but is not limited to, once every 3 weeks.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a capsid-modified recombinantadenovirus in accordance with embodiments of the disclosure.

FIG. 2 is a schematic diagram illustrating the construction of theHVR1-Modified Adenovirus DNA of an HVR1-modified recombinant adenovirusplasmid vector.

FIG. 3 is a schematic diagram illustrating the construction of theHVR5-Modified Adenovirus DNA of an HVR5-modified recombinant adenovirusplasmid vector.

FIG. 4 is a schematic diagram illustrating the construction of theFiber-Modified Adenovirus DNA of a Fiber-modified recombinant adenovirusplasmid vector.

FIG. 5 is a schematic diagram illustrating the construction of the HVR1and Fiber-Modified Adenovirus DNA of an HVR1 and Fiber-modifiedrecombinant adenovirus plasmid vector.

FIG. 6 is a schematic diagram illustrating the construction of the Fiberand pVII-Modified Adenovirus DNA of a Fiber and pVII-modifiedrecombinant adenovirus plasmid vector.

FIG. 7 is a schematic diagram illustrating the construction of the HVR1and pVII-Modified Adenovirus DNA of an HVR1 and pVII-modifiedrecombinant adenovirus plasmid vector.

FIG. 8 is a schematic diagram illustrating the construction of the HVR1,Fiber and pVII-Modified Adenovirus DNA of an HVR1, Fiber andpVII-modified recombinant adenovirus plasmid vector.

FIG. 9 is the nucleic acid sequence of codon-optimized Plasmodium yoeliicircumsporozoite protein (PyCS, SEQ ID NO:1) and the corresponding aminoacid sequence (SEQ ID NO:30)

FIG. 10 is the nucleic acid sequence of codon-optimized Plasmodiumfalciparum circumsporozoite protein (PfCSP, SEQ ID NO:2) and thecorresponding amino acid sequence (SEQ ID NO:43)

FIGS. 11A-C is the nucleic acid and amino acid sequences of a modifiedHexon having three repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=3) in HVR1 (SEQ ID NO:3, nucleic acid;SEQ ID NO:31, amino acid). The inserted (QGPGAP)₃ sequence isunderlined.

FIGS. 12A-C is the nucleic acid and amino acid sequences of a modifiedHexon having four repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=4) in HVR1 (SEQ ID NO:4, nucleic acid;SEQ ID NO: 32, amino acid). The inserted (QGPGAP)₄ sequence isunderlined.

FIGS. 13A-C is the nucleic acid and amino acid sequences of modifiedHexon having five repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=5) in HVR1 (SEQ ID NO:5, nucleic acid;SEQ ID NO: 33, amino acid). The inserted (QGPGAP)₅ sequence isunderlined.

FIGS. 14A-C is the nucleic acid and amino acid sequences of modifiedHexon having six repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=6) in HVR1 (SEQ ID NO:6, nucleic acid;SEQ ID NO: 34, amino acid). The inserted (QGPGAP)₆ sequence isunderlined.

FIGS. 15A-C is the nucleic acid and amino acid sequences of modifiedHexon having seven repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=7) in HVR1 (SEQ ID NO:7, nucleic acid;SEQ ID NO: 35, amino acid). The inserted (QGPGAP)₇ sequence isunderlined.

FIGS. 16A-C is the nucleic acid and amino acid sequences of modifiedHexon having eight repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=8) in HVR1 (SEQ ID NO:8, nucleic acid;SEQ ID NO: 36, amino acid). The inserted (QGPGAP)₈ sequence isunderlined.

FIGS. 17A-C is the nucleic acid and amino acid sequences of modifiedHexon having nine repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=9) in HVR1 (SEQ ID NO:9, nucleic acid;SEQ ID NO: 37, amino acid). The inserted (QGPGAP)₉ sequence isunderlined.

FIGS. 18A-C is the nucleic acid and amino acid sequences of modifiedHexon having eleven repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=11) in HVR1 (SEQ ID NO:10, nucleic acid;SEQ ID NO: 38, amino acid). The inserted (QGPGAP)₁₁ sequence isunderlined.

FIGS. 19A-C is the nucleic acid and amino acid sequences of modifiedHexon having twelve repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=12) in HVR1 (SEQ ID NO:11, nucleic acid;SEQ ID NO: 39, amino acid). The inserted (QGPGAP)₁₂ sequence isunderlined.

FIGS. 20A-C is the nucleic acid and amino acid sequences of modifiedHexon having four repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=4) in HVR1 (SEQ ID NO:12, nucleic acid; SEQID NO: 44, amino acid). The inserted (NANP)₄ sequence is underlined.

FIGS. 21A-C is the nucleic acid and amino acid sequences of modifiedHexon having six repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=6) in HVR1 (SEQ ID NO:13, nucleic acid; SEQID NO: 45, amino acid). The inserted (NANP)₆ sequence is underlined.

FIGS. 22A-C is the nucleic acid and amino acid sequences of modifiedHexon having eight repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=8) in HVR1 (SEQ ID NO:14, nucleic acid; SEQID NO: 46, amino acid). The inserted (NANP)₈ sequence is underlined.

FIGS. 23A-C is the nucleic acid and amino acid sequences of modifiedHexon having ten repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=10) in HVR1 (SEQ ID NO:15, nucleic acid;SEQ ID NO: 47, amino acid). The inserted (NANP)₁₀ sequence isunderlined.

FIGS. 24A-C is the nucleic acid and amino acid sequences of modifiedHexon having twelve repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=12) in HVR1 (SEQ ID NO:16, nucleic acid;SEQ ID NO: 48, amino acid). The inserted (NANP)₁₂ sequence isunderlined.

FIGS. 25A-C is the nucleic acid and amino acid sequences of modifiedHexon having fourteen repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=14) in HVR1 (SEQ ID NO:17, nucleic acid;SEQ ID NO: 49, amino acid). The inserted (NANP)₁₄ sequence isunderlined.

FIGS. 26A-C is the nucleic acid and amino acid sequences of modifiedHexon having sixteen repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=16) in HVR1 (SEQ ID NO:18, nucleic acid;SEQ ID NO: 50, amino acid). The inserted (NANP)₁₆ sequence isunderlined.

FIGS. 27A-C is the nucleic acid and amino acid sequences of modifiedHexon having eighteen repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=18) in HVR1 (SEQ ID NO:19, nucleic acid;SEQ ID NO: 51, amino acid). The inserted (NANP)₁₈ sequence isunderlined.

FIGS. 28A-C is the nucleic acid and amino acid sequences of modifiedHexon having twenty repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=20) in HVR1 (SEQ ID NO:20, nucleic acid;SEQ ID NO: 52, amino acid). The inserted (NANP)₂₀ sequence isunderlined.

FIGS. 29A-C is the nucleic acid and amino acid sequences of modifiedHexon having twenty-two repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=22) in HVR1 (SEQ ID NO:21, nucleic acid;SEQ ID NO: 53, amino acid). The inserted (NANP)₂₂ sequence isunderlined.

FIGS. 30A-C is the nucleic acid and amino acid sequences of modifiedHexon having twenty-eight repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=28) in HVR1 (SEQ ID NO:22, nucleic acid;SEQ ID NO: 54, amino acid). The inserted (NANP)₂₈ sequence isunderlined.

FIGS. 31A-C is the nucleic acid and amino acid sequences of modifiedHexon having three repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=3) in HVR5 (SEQ ID NO:23, nucleic acid;SEQ ID NO:40, amino acid). The inserted (QGPGAP)₃ sequence isunderlined.

FIGS. 32A-C is the nucleic acid and amino acid sequences of modifiedFiber having three repeats of the PyCS B cell epitope sequence(QGPGAP)_(n), (SEQ ID NO:59; n=3) in Fiber (SEQ ID NO:24, nucleic acid;SEQ ID NO:41, amino acid). The inserted (QGPGAP)₃ sequence isunderlined.

FIGS. 33A-B is the nucleic acid and amino acid sequences of modifiedFiber having four repeats of the PfCSP B cell epitope sequence(NANP)_(n), (SEQ ID NO:60; n=4) in Fiber (SEQ ID NO:25, nucleic acid;SEQ ID NO:55, amino acid). The inserted (NANP)₄ sequence is underlined.

FIG. 34 is the nucleic acid and amino acid sequences of the modifiedpVII having the PyCS CD4+ epitope sequence YNRNIVNRLLGDALNGKPEEK, (SEQID NO:61) at the N-terminus of pVII (SEQ ID NO:26, nucleic acid; SEQ IDNO:42, amino acid). The inserted YNRNIVNRLLGDALNGKPEEK sequence isunderlined.

FIG. 35 is the nucleic acid and amino acid sequences of the modifiedpVII having the PfCSP CD4+ epitope sequence EYLNKIQNSLSTEWSPCSVT, (SEQID NO:62) at the C-terminus of pVII (pVII-1; SEQ ID NO:27, nucleic acid;SEQ ID NO:56, amino acid). The inserted EYLNKIQNSLSTEWSPCSVT sequence isunderlined.

FIG. 36 is the nucleic acid and amino acid sequences of the modifiedpVII having the PfCSP CD4+ epitope sequence EYLNKIQNSLSTEWSPCSVT, (SEQID NO:62) before the first Nuclear Localization Signal (NLS) of pVII(pVII-2; SEQ ID NO:28, nucleic acid; SEQ ID NO:57, amino acid). Theinserted EYLNKIQNSLSTEWSPCSVT sequence is underlined.

FIG. 37 is the nucleic acid and amino acid sequences of the modifiedpVII having the PfCSP CD4+ epitope sequence EYLNKIQNSLSTEWSPCSVT, (SEQID NO:62) between the two NLSs of pVII (pVII-3; SEQ ID NO:29, nucleicacid; SEQ ID NO:58, amino acid). The inserted EYLNKIQNSLSTEWSPCSVTsequence is underlined.

FIG. 38 shows PyCS protein expression in AD293 cells after transienttransfection with PyCS-GFP/pShuttle-CMV. PyCS protein was detected bywestern blotting using mouse monoclonal anti-PyCS antibody (9D3).

FIG. 39 shows the results of silver staining and western blotting (A)and ELISA assay (B) of the purified capsid-modified recombinant PyCS-GFPadenoviruses to confirm the (QGPGAP)₃ epitope (SEQ ID NO:59; n=3)insertion into adenovirus capsid proteins. In the ELISA assay, ELISAplates were coated directly with purified adenoviruses and the insertedepitope in adenovirus particles was detected with anti-PyCS antibody.

FIG. 40 shows the results of silver staining and western blotting (A)and ELISA assay (B) of the purified capsid-modified recombinant PyCSadenoviruses to confirm the (QGPGAP)_(n) epitope (SEQ ID NO:59)insertion into adenovirus capsid proteins. In the ELISA assay, ELISAplates were coated directly with purified adenoviruses and the insertedepitope in adenovirus particles was detected with anti-PyCS antibody.

FIG. 41 illustrates a single immunization regimen with capsid-modifiedPyCS adenoviruses having (QGPGAP)_(n) repeats (SEQ ID NO:59, n=3, 4, 5,6, 9, 12) (A) and PyCS-specific CD8+ response two weeks afterimmunization (B).

FIG. 42 illustrates a prime and boost immunization regimen withcapsid-modified PyCS adenoviruses having (QGPGAP)_(n) repeats (SEQ IDNO:59, n=3) (A), PyCS-specific humoral responses at week 10 (B), andmalaria parasite burden in liver 42 hours after sporozoite challenge(C).

FIG. 43 illustrates anti-sporozoite antibody titer determined byindirect immunofluorescene assay (IFA) (A) and in vitro sporozoiteneutralizing activity (B) of pooled serum samples prepared from micegiven the regimen in FIG. 42.

FIG. 44 illustrates a prime and boost immunization regimen withcapsid-modified PyCS adenoviruses having (QGPGAP)_(n) repeats (SEQ IDNO:59, n=4, 6) in HVR1(A), PyCS-specific humoral responses at week 9(B), malaria parasite burden in liver 42 hours after sporozoitechallenge (C), and in vitro sporozoite neutralizing activity of pooledserum samples (D). Mice were immunized with or without adjuvant.

FIG. 45 illustrates a prime and boost immunization regimen withcapsid-modified PyCS adenoviruses having (QGPGAP)_(n) repeats (SEQ IDNO:59, n=6, 9, 12) in HVR1(A), PyCS-specific humoral responses at week 9(B), PyCS-specific CD8+ T cell responses at week 9 (C), and malariaparasite burden in liver 42 hours after sporozoite challenge (D). Micewere immunized with or without adjuvant.

FIG. 46 shows PfCSP protein expression in AD293 cells after transienttransfection with PfCSP/pShuttle-CMV. PfCSP was detected by westernblotting using mouse monoclonal anti-NANP antibody (2A10).

FIG. 47 illustrates the results of silver staining and western blotting(A), and ELISA assay (B) of the purified capsid-modified recombinantPfCSP adenoviruses to confirm the (NANP)₄ epitope (SEQ ID NO:60; n=4)insertion into adenovirus capsid proteins. The inserted (NANP)₄ epitope(SEQ ID NO:60; n=4) was detected with mouse monoclonal anti-NANPantibody (2A10). In the ELISA assay, ELISA plates were coated directlywith purified adenoviruses.

FIG. 48 shows the results of silver staining and western blotting (A)and ELISA assay (B) of the purified capsid-modified recombinant PfCSPadenoviruses to confirm the (NANP)_(n) epitope (SEQ ID NO:60; n=4, 6, 8,10, 12, 14, 16, 18, 20, 22) insertion into adenovirus capsid proteins.In the ELISA assay, ELISA plates were coated directly with purifiedadenoviruses and the inserted epitope in adenovirus particles wasdetected with anti-PfCSP antibody (2A10).

FIG. 49 illustrates the prime and boast immunization regimen withcapsid-modified recombinant PfCSP adenoviruses having (NANP)₄ (SEQ IDNO:60; n=4) (A) and PfCSP-specific humoral responses at week 9 (B).

FIG. 50 illustrates the prime and boast immunization regimen withcapsid-modified recombinant PfCSP adenoviruses having (NANP)_(n) (SEQ IDNO:60; n=4, 6, 8, 10) in HVR1 (A) and PfCSP-specific humoral responsesat week 9 (B).

FIG. 51 illustrates the prime and boast immunization regimen withcapsid-modified recombinant PfCSP adenoviruses having (NANP)_(n) (SEQ IDNO:60; n=10, 16, 22) in HVR1 (A) and PfCSP-specific humoral responses atweek 9 (B). Mice were immunized with or without adjuvant.

FIG. 52 illustrates the result of sliver staining analysis of purified(QGPGAP)₃-modified Fiber and pVII-1 ((QGPGAP)₃-Fib/CD4-pVII-1/PyCS-GFP)adenovirus (A) and anti-QGPGAP antibody titer at week 10 in miceimmunized with (QGPGAP)₃-Fib/PyCS-GFP or(QGPGAP)₃-Fib/CD4-pVII-1/PyCS-GFP as described in FIG. 49 (B). Theresults of two independent experiments were plotted in the figure afternormalization with the median antibody titers inB-Fib/PyCS-GFP-immunized group.

FIG. 53 shows schematic diagrams of the structure of the adenovirus pVIIproteins with the PfCSP CD4+ epitope sequence EYLNKIQNSLSTEWSPCSVT (SEQID NO:62) inserted at the before the first Nuclear Localization Signal(NLS) of pVII (PfCD4-pVII-2; SEQ ID NO:28, nucleic acid; SEQ ID NO:57,amino acid) and between the two NLSs of pVII (PfCD4-pVII-3; SEQ IDNO:29, nucleic acid; SEQ ID NO:58, amino acid) (A) and the results ofsilver staining to confirm the epitope insertion into pVII (B).

FIG. 54 illustrates the prime and boast immunization regimen with HVR1and pVII-modified recombinant PfCSP adenoviruses (A), PfCSP-specifichumoral responses at week 6 (B), and PfCSP-specific CD4+(EYLNKIQNSLSTEWSPCSVT; SEQ ID NO:62) response at week 9 (C).

FIG. 55 illustrates in vitro neutralization of recombinant adenovirus byhuman serum samples. AD293 cells were infected with recombinantadenoviruses in the presence of diluted human serum for overnight andGFP expression was measured as a marker of infection.

FIG. 56 illustrates the effect of anti-adenovirus immunity on theinduction of PyCS-specific T cell response by capsid-modified PyCS-GFPadenoviruses in vivo. (A) is the brief description of the study design.(B) shows PyCS-specific CD8+ T cell response in mice immunized withwild-type (wt)/empty adenovirus twice followed by priming withcapsid-modified PyCS-GFP adenoviruses.

FIG. 57 illustrates the effect of anti-adenovirus immunity on theinduction of PyCS-specific humoral immune response by capsid-modifiedPyCS-GFP adenoviruses in vivo. (A) is the brief description of the studydesign. (B) shows PyCS-specific humoral immune response in miceimmunized with wild-type (wt)/empty adenovirus twice followed by twodoses of capsid-modified PyCS-GFP adenoviruses.

MEANS FOR SOLVING THE PROBLEMS

The present inventors have found a novel recombinant adenovirus having anovel, capsid-modified structure that is derived from a recombinantadenovirus plasmid vector. The recombinant adenovirus is capable ofinfecting mammalian cells, causing the cells to express a Plasmodiumcircumsporozoite protein. The recombinant adenovirus also has one ormore capsid proteins that have been modified by having a desiredimmunogenic antigen, such as B cell epitope, T cell epitope ofPlasmodium circumsporozoite protein. The recombinant adenovirus isobtained by the method of transfecting cells with the linearizedrecombinant adenovirus plasmid vector. Using the obtained recombinantadenovirus, the present inventors carried out extensive research onpharmaceuticals containing as an active ingredient a recombinantadenovirus having malaria infection preventive and therapeutic effects.As a result, the inventors found that the obtained recombinantadenovirus has the desired pharmaceutical effects.

DETAILED DESCRIPTION

The following description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure.

The abbreviations used for the amino acids, peptides, base sequences,and nucleic acids in the present disclosure are based on theabbreviations specified in the IUPAC-IUB Communication on BiochemicalNomenclature, Eur. J. Biochem., 138: 9 (1984), “Guideline for PreparingSpecifications Including Base Sequences and Amino Acid Sequences”(United States Patent and Trademark Office), and those commonly used inthis technical field.

A “nucleotide sequence,” “polynucleotide” or “DNA molecule” ascontemplated by the current disclosure, may include double strand DNA orsingle strand DNA (i.e., a sense chain and an antisense chainconstituting the double strand DNA), and is not limited to a full lengththereof. Nucleotide sequences encoding an immunogenic foreign gene, suchas those disclosed herein below, encompass double strand DNA containinggenomic DNA, single strand DNA (sense chain) containing cDNA, singlestrand DNA (antisense chain) having a sequence complementary to thesense chain, synthetic DNA, and fragments thereof, unless otherwisementioned.

Nucleotide sequences, polynucleotides or DNA molecules as used hereinare not limited to the functional region, and may include at least oneof an expression suppression region, a coding region, a leader sequence,an exon, and an intron. Further, examples of nucleotide sequences orpolynucleotides may include RNA or DNA. A polypeptide containing aspecific amino acid sequence and a polynucleotide containing a specificDNA sequence may include fragments, homologs, derivatives, and mutantsof the polynucleotide. Examples of mutants of a nucleotide sequence orpolynucleotide (such as mutant DNA), include naturally occurring allelicmutants; artificial mutants; and mutants having deletion, substitution,addition, and/or insertion. It should be understood that such mutantsencode polypeptides having substantially the same function as thepolypeptide encoded by the original non-mutated polynucleotide.

The present disclosure relates to a recombinant adenovirus that canexpress an antigenic determinant of a Plasmodium parasite, and comprisesone or more modified capsid and/or core proteins. The recombinantadenovirus is derived from a recombinant adenovirus plasmid vector, thegeneration of which is described in the Examples below. The use ofadenovirus as a vector is discussed further below. The recombinantadenovirus plasmid vectors described herein may be used as a malariavaccine or pharmaceutical composition, wherein both humoral and cellularimmune responses against the Plasmodium parasite are induced.

The Plasmodium parasite may be selected from any of the known Plasmodium(P.) species, for example, P. falciparum, P. malariae, P. ovale, P.vivax, P. knowlesi, P. berghei, P. chabaudi and P. yoelii. In someembodiments, the antigenic determinant is derived from therodent-specific Plasmodium yoelii or the human-specific Plasmodiumfalciparum

In one embodiment, a recombinant adenovirus capsid-modified plasmidvector (also described as a recombinant adenovirus plasmid vectorherein) is a plasmid that encodes and produces a capsid and/orcore-modified recombinant adenovirus (also described as a recombinantadenovirus herein) that has a structure comprising one or more modifiedcapsid and/or core proteins. In accordance with the embodiments of thedisclosure, the modification of the capsid and/or core proteins may beaccomplished by insertion of at least one immunogenic epitope of aPlasmodium circumsporozoite protein. Alternatively, at least part of thecapsid and/or core protein may be deleted and replaced by at least oneimmunogenic epitope of a Plasmodium circumsporozoite protein. In someembodiments, the immunogenic epitope is a B-cell and/or T-cell epitopeof a Plasmodium circumsporozoite protein. The addition of a B cell or Tcell epitope may serve to enhance the efficacy of an adenoviral vectorused as a malaria vaccine by establishing or enhancing the humoralimmune response to the CS protein. The modified capsid and core proteinsand their significance with respect to their use in the recombinantadenovirus described herein are discussed further below.

The one or more modified capsid and/or core proteins may be a modifiedHexon protein, a modified Fiber protein, a modified pVII protein or acombination thereof. In one embodiment, a portion of a Hexonhypervariable region (HVR) and/or a portion of Fiber protein is replacedby at least one B-cell and/or T-cell epitope of a Plasmodiumcircumsporozoite protein. Alternatively, one or more B-cell and/or Tcell epitope of a Plasmodium circumsporozoite protein may be inserted inthe Fiber protein or Hexon HVR. In some aspects, the modified HVR may beHVR1, HVR2, HVR3, HVR4, HVR5, HVR6 or HVR7. In other aspects, themodified HVR may be HVR1 or HVR5. In some embodiments, the HVR-modifiedHexon may have a nucleic acid sequence of SEQ ID NO:3 (FIG. 11), SEQ IDNO:4 (FIG. 12), SEQ ID NO:5 (FIG. 13), SEQ ID NO:6 (FIG. 14), SEQ IDNO:7 (FIG. 15), SEQ ID NO:8 (FIG. 16), SEQ ID NO:9 (FIG. 17), SEQ IDNO:10 (FIG. 18), SEQ ID NO:11 (FIG. 19), SEQ ID NO:12 (FIG. 20), SEQ IDNO:13 (FIG. 21), SEQ ID NO:14 (FIG. 22), SEQ ID NO:15 (FIG. 23), SEQ IDNO:16 (FIG. 24), SEQ ID NO:17 (FIG. 25), SEQ ID NO:18 (FIG. 26), SEQ IDNO:19 (FIG. 27), SEQ ID NO:20 (FIG. 28), SEQ ID NO:21 (FIG. 29), SEQ IDNO:22 (FIG. 30), or SEQ ID NO:23 (FIG. 31). In other embodiments, themodified Fiber protein may have a nucleic acid sequence of SEQ ID NO:24(FIG. 32) or SEQ ID NO:25 (FIG. 33).

In another embodiment, a T-cell epitope of a Plasmodium circumsporozoiteprotein may be inserted into an adenovirus core pVII protein at any ofthe following sites: the C-terminus, before the first NuclearLocalization Signal (NLS) or between the two NLS. Alternatively, aT-cell epitope of a Plasmodium circumsporozoite protein may replace aportion of the pVII protein. In some embodiments, the modified pVIIprotein may have a nucleic acid sequence of SEQ ID NO:26 (FIG. 34), SEQID NO:27 (FIG. 35), SEQ ID NO:28 (FIG. 36) or SEQ ID NO:29 (FIG. 37).

In the recombinant adenovirus may express a transgenic protein orrecombinant transgenic protein. In some embodiments, the transgenicprotein or recombinant transgenic protein is a Plasmodiumcircumsporozoite protein or an antigenic determinant that is encoded bya recombinant adenovirus plasmid vector as described herein, and isexpressed by a recombinant adenovirus produced by said recombinantadenovirus plasmid vector after infection of one or more host cells,

Thus, in some embodiments, the recombinant adenovirus plasmid vectorscomprise a nucleotide sequence encoding a recombinant transgenicprotein. In one embodiment, the recombinant transgenic protein maycomprise an antigenic determinant of P. yoelii, a rodent-specificparasite, wherein the antigenic determinant comprises a P. yoeliicircumsporozoite (CS) protein gene or an antigenic portion thereof. Inanother embodiment, the recombinant transgenic protein may comprise anantigenic determinant of P. falciparum, a human-specific parasite,wherein the antigenic determinant comprises a P. falciparumcircumsporozoite gene (CS) protein or an antigenic portion thereof. TheP. falciparum CS protein has demonstrated prevention of malaria whenused as the basis of active immunization in humans againstmosquito-borne infection. The antigenic determinant may further comprisean immunogenic epitope, such as a B cell and/or T cell epitope.

In some embodiments, the CS protein is codon-optimized for enhancedexpression in a subject. Codon-optimization is based on the requiredamino acid content, the general optimal codon usage in the subject ofinterest as well as any aspects that should be avoided to ensure properexpression. Such aspects may be splice donor or acceptor sites, stopcodons, polyadenylation (pA) signals, GC- and AT-rich sequences,internal TATA boxes, or any other aspects known in the art. In someembodiments, the DNA sequence of the codon-optimized CS transgene isshown in FIG. 9 (SEQ ID NO:1, P. yoelii) and FIG. 10 (SEQ ID NO: 2, P.falciparum).

In some embodiments, the recombinant adenovirus plasmid vector may beone of the following modified P. falciparum recombinant adenovirusplasmid vectors: HVR1-modified adenovirus vector (NANP-HVR1/PfCSP)constructed as shown in FIG. 2, using a B cell epitope coding sequenceof (NANP)_(n) (SEQ ID NO:60); Fiber-modified adenovirus vector(NANP-Fib/PfCSP) constructed as shown in FIG. 4, using a B cell epitopecoding sequence of (NANP)_(n) (SEQ ID NO:60); HVR1 and Fiber-modifiedadenovirus vector (NANP-HVR1/B-Fib/PfCSP) constructed as shown in FIG.5, using a B cell epitope coding sequence of (NANP)_(n) (SEQ ID NO:60);HVR1 and pVII-modified adenovirus vector (NANP-HVR1/CD4-pVII/PfCSP)constructed as shown in FIG. 7, using a B cell epitope of (NANP)_(n)(SEQ ID NO:60) and a CD4 epitope coding sequence of EYLNKIQNSLSTEWSPCSVT(SEQ ID NO:62); Fiber and pVII-modified adenovirus vector(NANP-Fib/CD4-pVII/PfCSP) constructed as shown in FIG. 6, using a B cellepitope of (NANP)_(n) (SEQ ID NO:60) and a CD4 epitope coding sequenceof EYLNKIQNSLSTEWSPCSVT (SEQ ID NO:62); and HVR1, Fiber andpVII-modified adenovirus vector (NANP-HVR1/Fib/CD4-pVII/PfCSP)constructed as shown in FIG. 8, using a B cell epitope of (NANP)_(n)(SEQ ID NO:60) and a CD4 epitope coding sequence of EYLNKIQNSLSTEWSPCSVT(SEQ ID NO:62).

In other embodiments, the recombinant adenovirus plasmid vector may beone of the following modified P. yoelii recombinant adenovirus plasmidvectors: HVR1-modified adenovirus vector (QGPGAP-HVR1/PyCS) constructedas shown in FIG. 2, using a B cell epitope coding sequence of(QGPGAP)_(n) (SEQ ID NO:59); Fiber-modified adenovirus vector(QGPGAP-Fib/PyCS) constructed as shown in FIG. 4, using a B cell epitopecoding sequence of (QGPGAP)_(n) (SEQ ID NO:59); HVR1 and Fiber-modifiedadenovirus vector (QGPGAP-HVR1/B-Fib/PyCS) constructed as shown in FIG.5, using a B cell epitope coding sequence of (QGPGAP)_(n) (SEQ IDNO:59); HVR1 and pVII-modified adenovirus vector(QGPGAP-HVR1/CD4-pVII/PyCS) constructed as shown in FIG. 7, using a Bcell epitope of (QGPGAP)_(n) (SEQ ID NO:59) and a CD4 epitope codingsequence of YNRNIVNRLLGDALNGKPEEK, (SEQ ID NO:61); Fiber andpVII-modified adenovirus vector (QGPGAP-Fib/CD4-pVII/PyCS) constructedas shown in FIG. 6, using a B cell epitope of (QGPGAP)_(n) (SEQ IDNO:59) and a CD4 epitope coding sequence of YNRNIVNRLLGDALNGKPEEK, (SEQID NO:61); and HVR1, Fiber and pVII-modified adenovirus vector(QGPGAP-HVR1/Fib/CD4-pVII/PyCS) constructed as shown in FIG. 8, using aB cell epitope of (QGPGAP)_(n) (SEQ ID NO:59) and a CD4 epitope codingsequence of YNRNIVNRLLGDALNGKPEEK, (SEQ ID NO:61).

In other embodiments, a recombinant adenovirus may be produced by one ofthe following modified P. falciparum or P. yoelii recombinant adenovirusplasmid vectors: NANP-HVR1/PfCSP or QGPGAP-HVR1/PyCS (FIG. 2),NANP-Fib/PfCSP or QGPGAP-Fib/PyCS (FIG. 4), NANP-HVR1/B-Fib/PfCSP orQGPGAP-HVR1/B-Fib/PyCS (FIG. 5), NANP-HVR1/CD4-pVII/PfCSP orQGPGAP-HVR1/CD4-pVII/PyCS (FIG. 7), NANP-Fib/CD4-pVII/PfCSP orQGPGAP-Fib/CD4-pVII/PyCS (FIG. 6), NANP-HVR1/Fib/CD4-pVII/PfCSP orQGPGAP-HVR1/Fib/CD4-pVII/PyCS (FIG. 8). The recombinant adenovirus maybe produced in accordance with the methods described herein forproducing a recombinant adenovirus plasmid vector with the ability toexpress a recombinant transgenic protein (e.g., Plasmodium CS protein)in mammalian host cells.

Purification of a recombinant adenovirus may be performed by using knownvirus purification methods. For example, purification of 0.5 to 1.0 mLof a stock virus obtained by the method of producing a recombinantadenovirus protein by inoculating insect cells (1×10⁷ cells/10 cm dish),such as AD293 cells. The culture supernatant is then collected severaldays after the infection, and a virus pellet obtained by centrifugationis suspended in a buffer, such as PBS (Phosphate Buffered Saline). Theresulting suspension is subjected to a sucrose gradient of 10 to 60% andthen centrifuged (25,000 rpm for 60 minutes at 4° C.) to collect a virusband. The collected virus is further suspended in PBS, subsequentlycentrifuged under the same conditions as above, and the resultingpurified recombinant virus pellet is stored at 4° C. in a buffer, suchas PBS.

Another embodiment is directed to a pharmaceutical compositionessentially comprising at least one active ingredient. In oneembodiment, an active ingredient of the pharmaceutical composition maycomprise a recombinant adenovirus, which may be obtained by the geneticengineering techniques described herein. More specifically, the activeingredient may be a recombinant adenovirus comprising modified capsidand/or core proteins, wherein a portion of a Hexon hypervariable region(HVR), a portion of Fiber protein, a portion of pVII protein or acombination thereof is replaced by at least one immunogenic epitope ofPlasmodium circumsporozoite protein. Alternatively, one or more B-celland/or T cell epitope of a Plasmodium circumsporozoite protein may beinserted in the Fiber protein, Hexon HVR or pVII protein. Therecombinant adenovirus plasmid vector further comprises a transgenicprotein or recombinant transgenic protein that is expressed by therecombinant adenovirus after infecting one or more host cells. Thetransgenic protein or recombinant transgenic protein may be a Plasmodiumcircumsporozoite protein or a malaria antigen of a Plasmodiumcircumsporozoite protein, wherein the malaria antigen comprises at leastone immunogenic epitope (e.g., a B cell or T cell epitope) of Plasmodiumcircumsporozoite protein.

In some embodiments, the active ingredient of the pharmaceuticalcomposition is a recombinant adenovirus derived from a recombinantadenovirus plasmid vector, wherein the recombinant adenovirus plasmidvector is one of the following modified P. falciparum or P. yoeliirecombinant adenovirus plasmid vectors: NANP-HVR1/PfCSP orQGPGAP-HVR1/PyCS (FIG. 2), NANP-Fib/PfCSP or QGPGAP-Fib/PyCS (FIG. 4),NANP-HVR1/B-Fib/PfCSP or QGPGAP-HVR1/B-Fib/PyCS (FIG. 5),NANP-HVR1/CD4-pVII/PfCSP or QGPGAP-HVR1/CD4-pVII/PyCS (FIG. 7),NANP-Fib/CD4-pVII/PfCSP or QGPGAP-Fib/CD4-pVII/PyCS (FIG. 6),NANP-HVR1/Fib/CD4-pVII/PfCSP or QGPGAP-HVR1/Fib/CD4-pVII/PyCS (FIG. 8).These recombinant adenovirus plasmid vectors are capable of producingrecombinant adenoviruses when transfected into cells (e.g., AD293 cells)and wherein the recombinant transgenic protein may be expressed inmammalian cells, including human cells.

When given to a subject, a pharmaceutical composition having an activeingredient is a recombinant adenovirus as described herein enhancesmalaria infection-preventing effects against a malaria infectiousantigen and reduces the infectivity titer, as described further in theExamples below. Thus, the recombinant adenovirus may be used for thetreatment of malaria infections associated with infection of targetcells and tissues. Examples of target cells affected by such malariainfection include blood cells, hepatic cells, renal cells, brain cells,lung cells, epithelial cells, and muscular cells. Examples of tissuescomprising such cells include the lung, liver, kidney, brain, arteriesand veins, the stomach, intestines, urethra, skin, and muscle.

In some aspects, the pharmaceutical composition may enhance malariainfection-preventing effects against infectious antigens, for example,malaria antigens such as sporozoite surface antigens (CircumsporozoiteProtein (CSP) and Thrombospondin Related Adhesive Protein (TRAP)) ofmalaria parasites, merozoite surface membrane protein (MSPI), malaria Santigen secreted from erythrocytes infected with malaria, and P.falciparum Erythrocyte Membrane Protein-1 (PfEMPI) protein present inthe knobs of erythrocytes infected with malaria. The pharmaceuticalcomposition may enhance malaria infection-preventing effects against aPlasmodium parasite, selected from any known Plasmodium (P) species, forexample, P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi, P.berghei, P. chabaudi and P. yoelii, by reducing the infectivity titer.When administered to a subject, a reduction of the infectivity titer bythe pharmaceutical composition may result in an increased survival,disease-free survival, or infection-free survival period and survival,disease-free survival, or infection-free survival rate when compared tosubjects not administered the pharmaceutical composition. Thus, in someaspects, the pharmaceutical composition is useful as a preventive ortherapeutic agent for malaria infections caused by pathogens such asPlasmodium. In further aspects, the pharmaceutical composition is usefulas a preventive or therapeutic agent for complications resulting from amalaria infection caused by pathogens such as Plasmodium.

The infection-preventing effect of the recombinant adenovirus of thepresent invention in a subject can be provided, for example, byadministering the pharmaceutical composition containing thecapsid-modified recombinant adenovirus of the present invention andadditives for pharmaceutical administration to vertebrates, particularlymammals, including humans, by intramuscular (i.m.), subcutaneous (s.c.),intracutaneous (i.c.), intradermal (i.d.), intraperitoneal (i.p.),nasal, or respiratory route, and then immunizing the vertebrates withthe pharmaceutical composition containing the recombinant adenovirusdescribed herein as an active ingredient several times. To evaluate theinfection-preventing effect, the survival rate, disease-free survival,or infection-free survival of subjects immunized with the pharmaceuticalcomposition several times followed by infection by a target pathogen(such as a selected Plasmodium species) may be compared with thesurvival rate, disease-free survival, or infection-free survival ofsubjects not given the pharmaceutical composition.

In some embodiments, the pharmaceutical composition may additionallycomprise a pharmaceutically effective amount of capsid and/orcore-modified recombinant adenovirus as described herein and apharmaceutically acceptable carrier, which is described further below.

Another embodiment is directed to a vaccine composition essentiallycomprising at least one active ingredient. In one embodiment, an activeingredient of the vaccine composition may comprise a recombinantadenovirus, derived from a recombinant adenovirus plasmid vector asdescribed herein. More specifically, the active ingredient may be arecombinant adenovirus comprising modified capsid or core proteins,wherein a portion of a Hexon hypervariable region (HVR), a portion ofFiber protein, a portion of pVII protein or a combination thereof arereplaced by at least one immunogenic epitope of Plasmodiumcircumsporozoite protein. Alternatively, at least one immunogenicepitope of a Plasmodium circumsporozoite protein may be inserted in thepVII protein, Fiber protein or Hexon HVR, or a combination thereof. Insome embodiments, the active ingredient of the vaccine composition maybe derived from a recombinant adenovirus plasmid vector illustrated inFIGS. 2-8, for example, NANP-HVR1/PfCSP or QGPGAP-HVR1/PyCS (FIG. 2),NANP-Fib/PfCSP or QGPGAP-Fib/PyCS (FIG. 4), NANP-HVR1/B-Fib/PfCSP orQGPGAP-HVR1/B-Fib/PyCS (FIG. 5), NANP-HVR1/CD4-pVII/PfCSP orQGPGAP-HVR1/CD4-pVII/PyCS (FIG. 7), NANP-Fib/CD4-pVII/PfCSP orQGPGAP-Fib/CD4-pVII/PyCS (FIG. 6), NANP-HVR1/Fib/CD4-pVII/PfCSP orQGPGAP-HVR1/Fib/CD4-pVII/PyCS (FIG. 8).

In some aspects, the vaccine composition, when administered to asubject, first comprises a recombinant adenovirus having one or moreantigenic portions of a Plasmodium CS protein (i.e., a B cell epitope, Tcell epitope or both) inserted into or replacing at least a part of acapsid or core protein. The vaccine composition may then express arecombinant transgenic protein, wherein the recombinant transgenicprotein is a Plasmodium CS protein comprising a B cell epitope, T cellepitope or both. The antigenic portions of the Plasmodium CS protein arefound in the recombinant transgenic protein and the modified capsid orcore proteins promote or enhance acquired humoral immunity, cellularimmunity, or both as described in the Examples below. Thus, in someaspects, the recombinant adenovirus as described herein is useful as avaccine to promote or enhance humoral immunity, cellular immunity, orboth.

In further embodiments, the vaccine composition may enhanceinfection-preventing effects against infectious antigens, for example,malaria antigens such as sporozoite surface antigens (CSP and TRAP) ofmalaria parasites, merozoite surface membrane protein MSPI, malaria Santigen secreted from erythrocytes infected with malaria, PfEMPI proteinpresent in the knobs of erythrocytes infected with malaria, Serine-RichAntigen (SERA) protein, Tyrosine-Rich Acidic Matrix Protein (TRAMP), andApical Membrane Antigen-1 (AMAI) protein. Further, a reduced infectivitytiter (e.g., the viral infectivity titer) resulting from administrationof a vaccine composition described herein may result in an increasedsurvival, disease-free survival or infection-free survival period andsurvival, disease-free survival or infection-free survival rate whencompared to subjects not administered the vaccine composition. Thus, insome aspects, the vaccine composition is also useful as a preventive ortherapeutic agent for malaria infections caused by pathogens such asPlasmodium. In further aspects, the vaccine composition is also usefulas a preventive or therapeutic agent for complications resulting from amalaria infection by pathogens such as Plasmodium.

A vaccine composition as described herein may comprise a therapeuticallyeffective amount of a recombinant adenovirus as described herein, andfurther comprising a pharmaceutically acceptable carrier according to astandard method. Examples of acceptable carriers include physiologicallyacceptable solutions, such as sterile saline and sterile bufferedsaline.

In some embodiments, the vaccine or pharmaceutical composition may beused in combination with a pharmaceutically effective amount of anadjuvant to enhance the anti-malaria effects. Any immunologic adjuvantthat may stimulate the immune system and increase the response to avaccine, without having any specific antigenic effect itself may be usedas the adjuvant. Many immunologic adjuvants mimic evolutionarilyconserved molecules known as pathogen-associated molecular patterns(PAMPs) and are recognized by a set of immune receptors known asToll-like Receptors (TLRs). Examples of adjuvants that may be used inaccordance with the embodiments described herein include Freund'scomplete adjuvant, Freund's incomplete adjuvant, double stranded RNA (aTLR3 ligand), LPS, LPS analogs such as monophosphoryl lipid A (MPL) (aTLR4 ligand), flagellin (a TLR5 ligand), lipoproteins, lipopeptides,single stranded RNA, single stranded DNA, imidazoquinolin analogs (TLR7and TLR8 ligands), CpG DNA (a TLR9 ligand), Ribi's adjuvant(monophosphoryl-lipid A/trehalose dicorynoycolate), glycolipids(α-GalCer analogs), unmethylated CpG islands, oil emulsion, liposomes,virosomes, saponins (active fractions of saponin such as QS21), muramyldipeptide, alum, aluminum hydroxide, squalene, BCG, cytokines such asGM-CSF and IL-12, chemokines such as MIP 1-α and RANTES,N-acetylmuramine-L-alanyl-D-isoglutamine (MDP), thymosin αI and MF59.The amount of adjuvant used can be suitably selected according to thedegree of symptoms, such as softening of the skin, pain, erythema,fever, headache, and muscular pain, which might be expressed as part ofthe immune response in humans or animals after the administration ofthis type of vaccine.

In some embodiments, the vaccine or pharmaceutical composition describedherein may be used in combination with other known pharmaceuticalproducts, such as immune response-promoting peptides and antibacterialagents (synthetic antibacterial agents). The vaccine or pharmaceuticalcomposition may further comprise other drugs and additives. Examples ofdrugs or additives that may be used in conjunction with a vaccine orpharmaceutical composition described herein include drugs that aidintracellular uptake of the recombinant adenovirus or recombinanttransgenic protein of the present invention, liposome and other drugsand/or additives that facilitate transfection, (e.g., fluorocarbonemulsifiers, cochleates, tubules, golden particles, biodegradablemicrospheres, and cationic polymers).

In some embodiments, the amount of the active ingredient contained inthe vaccine or pharmaceutical composition described herein may beselected from a wide range of concentrations, Virus Particle Unit (VPU),Plaque Forming Unit (PFU), weight to volume percent (w/v %) or otherquantitative measure of active ingredient amount, as long as it is atherapeutically or pharmaceutically effective amount. The dosage of thevaccine or pharmaceutical composition may be appropriately selected froma wide range according to the desired therapeutic effect, theadministration method (administration route), the therapeutic period,the patient's age, gender, and other conditions, etc.

In some aspects, when a recombinant adenovirus is administered to ahuman subject as an active ingredient of the vaccine or pharmaceuticalcomposition, the dosage of the recombinant adenovirus may beadministered in an amount approximately corresponding to 10² to 10¹⁴PFU, preferably 10⁵ to 10¹² PFU, and more preferably 10⁶ to 10¹⁰ PFU perpatient, calculated as the PFU of the recombinant virus.

In further aspects, when a recombinant adenovirus is administered to asubject as an active ingredient of the vaccine or pharmaceuticalcomposition, the dosage may be selected from a wide range in terms ofthe amount of expressible DNA introduced into the vaccine host or theamount of transcribed RNA. The dosage also depends on the strength ofthe transcription and translation promoters used in any transfer vectorsused.

In some embodiments, the vaccine composition or pharmaceuticalcomposition described herein may be administered by directly injecting arecombinant adenovirus suspension prepared by suspending the recombinantadenovirus in PBS (phosphate buffered saline) or saline into a localsite (e.g., into the lung tissue, liver, muscle or brain), by nasal orrespiratory inhalation, or by intravascular (i.v.) (e.g.,intra-arterial, intravenous, and portal venous), subcutaneous (s.c.),intracutaneous (i.c.), intradermal (i.d.), or intraperitoneal (i.p.)administration. The vaccine or pharmaceutical composition of the presentinvention may be administered more than once. More specifically, afterthe initial administration, one or more additional vaccinations may begiven as a booster. One or more booster administrations can enhance thedesired effect. After the administration of the vaccine orpharmaceutical composition, booster immunization with a pharmaceuticalcomposition containing the recombinant adenovirus as described hereinmay be performed.

In further embodiments, use of various other adjuvants, drugs oradditives with the vaccine of the invention, as discussed above, mayenhance the therapeutic effect achieved by the administration of thevaccine or pharmaceutical composition. The pharmaceutically acceptablecarrier may contain a trace amount of additives, such as substances thatenhance the isotonicity and chemical stability. Such additives should benon-toxic to a human or other mammalian subject in the dosage andconcentration used, and examples thereof include buffers such asphosphoric acid, citric acid, succinic acid, acetic acid, and otherorganic acids, and salts thereof; antioxidants such as ascorbic acid;low molecular weight (e.g., less than about 10 residues) polypeptides(e.g., polyarginine and tripeptide) proteins (e.g., serum albumin,gelatin, and immunoglobulin); amino acids (e.g., glycine, glutamic acid,aspartic acid, and arginine); monosaccharides, disaccharides, and othercarbohydrates (e.g., cellulose and derivatives thereof, glucose,mannose, and dextrin), chelating agents (e.g., EDTA); sugar alcohols(e.g., mannitol and sorbitol); counterions (e.g., sodium); nonionicsurfactants (e.g., polysorbate and poloxamer); and PEG.

The vaccine or pharmaceutical composition containing a recombinantadenovirus described herein may be stored as an aqueous solution or alyophilized product in a unit or multiple dose container such as asealed ampoule or a vial. Another embodiment further provides a methodof preventing malaria infection, or a method of treating malariacomprising administering an effective amount of the recombinantadenoviral vaccine, formulation, or pharmaceutical composition.

The present invention further provides a method of immunostimulationcomprising administering an effective amount of a recombinant adenoviralvaccine composition, formulation, pharmaceutical composition or acombination thereof to a subject. Subjects may include humans, animals(such as mammals, birds, reptiles, fish, and amphibians), or any othersubjects that may become infected with a malaria parasite. Malariaparasites may include a Plasmodium parasite, selected from any of knownPlasmodium (P) species, for example, P. falciparum, P. malariae, P.ovale, P. vivax, P. knowlesi, P. berghei, P. chabaudi and P. yoelii.

In some embodiments, a recombinant adenovirus as described herein may beformed alone or may be together with a pharmaceutically acceptablecarrier into a vaccine composition, formulation, or pharmaceuticalcomposition, and administered to the subject. The administration routemay be, for example, any administration route mentioned above. Thepharmaceutically acceptable carrier for use in the present invention canbe suitably selected from carriers commonly used in this technicalfield, according to the form of the pharmaceutical composition to beproduced. For example, when the pharmacological composition is formedinto an aqueous solution, purified water (sterile water) or aphysiological buffer solution can be used as the carrier. When thepharmaceutical composition is formed into other appropriate solutions,organic esters capable of being injected, such as glycol, glycerol andolive oil may be used as the carrier. The composition may containstabilizers, excipients and other commonly used substances in thistechnical field, and particularly in the field of vaccine formulations.

In further embodiments, the amount of recombinant adenovirus used in avaccine composition, formulation, or pharmaceutical composition may besuitably selected from a wide range of concentrations, VPU, PFU, weightto volume percent (w/v %) or other quantitative measure of activeingredient amount. In some aspects, a suitable range of recombinantadenovirus in the composition is preferably about 0.0002 to about 0.2(w/v %), and more preferably 0.001 to 0.1 (w/v %). The method ofadministration of a recombinant adenovirus vaccine composition,formulation, or pharmaceutical composition according to some embodimentsmay be suitably selected according to the dosage form, the patient'sage, gender and other conditions such as the severity of the disease. Asuitable dosage form is a form for parenteral administration, such asinjections, drops, nasal drops, and inhalants. When the composition isformed into an injection or drops, the injection can be intravenouslyadministered and mixed with a replacement fluid such as a glucosesolution or an amino acid solution as appropriate, or can beadministered intramuscularly (i.m.), intracutaneously (i.c.),subcutaneously (s.c.) intradermally (i.d.), or intraperitoneally (i.p.).

In other embodiments, the daily dosage of a recombinant adenovirusvaccine composition, formulation, or pharmaceutical composition may varydepending on the subject's condition, body weight, age, gender, etc. Insome aspects, the dosage of a recombinant adenovirus is administered inan amount of approximately 0.001 to 100 mg per kg of body weight perday. The vaccine, formulation, or composition of the invention may beadministered in one or more administrations per day.

In further embodiments, when a recombinant adenovirus is administered toa human subject as an active ingredient of the vaccine composition,formulation or pharmaceutical composition, the dosage of the recombinantadenovirus is administered in an amount approximately corresponding to10² to 10¹⁴ PFU, preferably 10⁵ to 10¹² PFU, and more preferably 10⁶ to10¹⁰ PFU per patient, calculated as the PFU of the recombinantadenovirus particle. The vaccine composition of the present inventionshould be administered according to Good Medical Practice, consideringthe clinical condition (for example, the condition to be prevented ortreated) of each patient, the delivery site of the vaccine compositioncontaining the recombinant adenovirus, the target tissue, theadministration method, the dosage regimen, and other factors known tothose skilled in the art. Therefore, the proper dosage of the vaccinecomposition herein is determined in consideration of the above.

Yet another embodiment of the disclosure relates to a method of treatingor preventing a malaria infection in a subject, the method comprisingadministering an immunologic or therapeutic amount of a malaria vaccinecomposition comprising a recombinant adenovirus. The recombinantadenovirus of the malaria vaccine may comprise an antigenic determinantof a Plasmodium parasite, and may further comprise one or more modifiedcapsid or core proteins. An immunologic, pharmacologic or therapeuticamount may be any suitable amount wherein a potent immune response isgenerated against one or more antigenic portions of the (CS) protein(i.e., the transgene, B cell epitope, or CD4+ T cell epitope) such thatmalarial infection is prevented or reduced in severity.

When a subject is first exposed or “primed” to an adenovirus vector, theimmune system produces neutralizing antibodies against that specificvector. The immune response to the adenovirus is generally directedagainst the capsid proteins. Therefore, subsequent exposure to the sameadenovirus vector, or “boosts,” can reduce the efficacy of transgeneexpression. Therefore, in some embodiments, the method of treating orpreventing a malaria infection described above may comprise a primingstep using a first recombinant adenovirus vector followed by one or moreboosting steps using one or more different recombinant adenovirusvectors. This method may be used in subjects that have not yet beenexposed to a wild-type adenovirus, or in a subject that has beenpreviously exposed to a wild-type adenovirus vector, wherein the primingstep recombinant adenovirus vector is used to circumvent existingadenovirus immunity. Further embodiments and examples are describedbelow.

Adenovirus as a Vector

Adenoviruses are non-enveloped DNA viruses comprising a set of viralcapsid proteins (described below) and a viral genome, that have beenwidely used to deliver one or more therapeutic or antigenic transgene toa variety of cells in vitro and in vivo. Many adenovirus serotypesexist. Of the known adenovirus serotypes, serotype 5 (Ad5) is preferablyused as a vector for foreign gene transduction because of its stronginfectivity in vivo (Abbink et al. 2007). Expression of the antigenictransgene may be controlled by any promoter or enhancer element known inthe art. Promoters which may be used to control gene expression include,but are not limited to, cytomegalovirus immediate early promoter (CMV),simian virus 40 (SV40) early promoter, cellular polypeptide chainelongation factor 1 alpha (EF1) promoter, Rous sarcoma virus (RSV)promoter, and tetracycline-regulated (TR) promoter. A polyadenylation(pA) signal after the coding sequence may also be used for efficienttranscription and translation. The recombinant adenovirus vectordescribed herein may be replication-defective, having a deletion atleast in the E1 region of the adenoviral genome, since the E1 region isrequired for replication, transcription, translation and packagingprocesses. In some aspects, the E2, E3 and/or E4 regions may also bedeleted. In further aspects, a Kozak consensus sequence may be used fora more efficient translation (Kozak 1987).

The adenovirus (Ad) system is an attractive vector for the developmentof recombinant vaccines for a number of reasons. One reason is thatrecombinant adenoviral vectors infect most mammalian cell types (bothreplicative and non-replicative), including, but not limited to, mouseand human cell types. Thus, the same vector may be used successfully inmouse models and human clinical trials alike. Another reason is that anytransferred genetic information remains epichromosomal, avoidinginsertional mutagenesis and alteration of the cellular genotype (Crystal1995). Yet another reason is that the transgene remains unaltered aftersuccessive rounds of viral replication. Other advantages of usingadenovirus include that recombinant adenovirus: 1) has a high virionstability, 2) is well tolerated, 3) may be grown at high titer, 4) canaccommodate large transgenes, 5) has a genome that has been extensivelystudied for many years such that the complete DNA sequence of severalserotypes is known, facilitating the manipulation of the Ad genome byrecombinant DNA techniques (Graham and Prevec 1992).

In one embodiment, the adenovirus vaccine platform is used as a viralvector for development of a vaccine that targets a pre-erythrocyticmalaria parasite, and provides protection from malaria infection. Amongknown recombinant viral vectors (Rodrigues et al. 1997, Bruña-Romero etal. 2001, Anderson et al. 2004, Tao et al. 2005), adenovirus has beenshown to be a suitable viral vector for a malaria vaccine because it caninduce a strong protective cellular immune response to pre-erythrocyticmalaria parasites (Rodrigues et al. 1997). The malaria parasite may beany one of the Plasmodium family. In some embodiments, the targetedparasite may be P. yoelii or P. falciparum.

Adenovirus Vectors Expressing PyCS as a Transgene Elicits aMalaria-Specific CD8+ T Cell Response

Adenovirus is an attractive vector for inducing a significant CD8+ Tcell-mediated protective immunity against malaria (Rodrigues et al.1997, Rodrigues et al. 1998). The immunogenicity of a recombinantadenovirus expressing the P. yoelii (a rodent malaria parasite) CSprotein, AdPyCS, was determined using a rodent malaria model. Theinoculation of mice with AdPyCS induces complete immunity in asignificant proportion of mice, preventing the occurrence of parasitemia(Rodrigues et al. 1997). This protective effect is primarily mediated byCD8+ T cells, as evidenced by depletion of the T cell population and iscorroborated by the fact that AdPyCS was unable to induce high titers ofantibody response against malaria parasites.

To quantitatively measure the infectivity of capsid-modified adenovirus,the shuttle vector may contain a GFP expression cassette and cloningsites for a transgene. The resulting shuttle vector (GFP/pShuttle-CMV)has dual pCMV promoters and SV40pAs for a transgene and GFP from pmaxGFP(Amaxa, Germany). The optimized PyCS fragment was inserted into KpnI andHindIII sites of GFP/pShuttle-CMV.

The immunogenicity of Ad(PyCS+GFP) was determined by measuring themagnitude of the CS-specific CD8+ T cell response and the level ofprotective immunity against the plasmodial liver stages. Administrationof Ad(PyCS+GFP) via different routes, at an optimal dose, 10¹⁰ viralparticle (v.p.) elicited the same pattern of anti-malarial protectiveresponses that AdPyCS was shown to elicit, with the s.c. and i.m. routesinducing the strongest response resulted in the highest degree of liverstage inhibition in mice challenged with live P. yoelii sporozoites.This illustrates that as a vaccine, Ad(PyCS+GFP) behaves equivalently toAdPyCS (Rodrigues et al 1997), and is a potentially useful tool indetermining the in vivo tropism of AdPyCS.

Adenovirus Capsid and Core Proteins

The studies above confirm that recombinant adenoviral vectors expressinga CS protein elicit a strong cellular immune response by CD8+ T cells,but no appreciable humoral response. Therefore, because the humoralresponse to wild-type adenovirus can often be attributed to capsidproteins, recombinant adenoviral vectors with modified capsid and coreproteins were constructed to 1) enhance humoral immunity via B cellactivation, 2) enhance humoral immunity via T helper cell activation,and 3) circumvent existing adenoviral immunity.

Adenovirus is a non-enveloped naked double stranded DNA virus with anicosahedral shape, having 20 faces of equilateral triangles. Theadenovirus capsid consists of 252 capsomers, of which 240 are Hexontrimers and 12 are penton pentamers. A Fiber protein, which projectsfrom each penton base, mediates attachment to host cells by interactionwith the cellular receptor. A secondary interaction occurs between theRGD (Asp-Arg-Gly) motif in the penton base with αvβ3, αvβ5 and similarintegrins, facilitating subsequent internalization of adenovirus intothe cell (Mathias et al. 1994, Wickham et al. 1993). Most of theadenovirus use the coxsackie-adenovirus receptor, CAR, as a cellularreceptor (Bergelson et al. 1997). In addition, MHC class I molecules,VCAM, and heparan sulfate, are shown to mediate attachment and entry ofAd5 (Chu et al. 2001, Hong et al. 1997). Following entry viaendocytosis, the Ad5 rapidly escapes from endocytic compartments intothe cytosol (Meier and Greber 2003, Leopold and Crystal 2007). Thevirion then translocates to the nucleus using microtubules. The Fiberprotein is shed as the earliest capsid protein in the process (Nakano etal. 2000, Hong et al 2003). Adenoviruses of different serotypesdemonstrate different trafficking patterns (Miyazawa et al. 1999,Miyazawa et al. 2001). Changing or modifying the Fiber protein canimpact trafficking, which may be particularly important with regard toantigen processing and presentation, following infection of antigenpresenting cells (APC).

The adenovirus Fiber is a trimer divided into Fiber tail, shaft and knobdomains (Henry et al. 1994, Rux and Burnett 2004, Chroboczek et al.1995). The three dimensional structure of the knob domain is known, andtogether with mutagenesis studies, these studies allow the areasinvolved in CAR interaction and trimerization to be visualized (Kirby etal. 1999, Xia et al. 1995). The Fiber shaft projects from the virion andthe Fiber knob contains the Coxsackie and Adenovirus Receptor (CAR)interaction domain (Roelvink et al. 1999, Bewley et al. 1999). TheCAR-binding site of the Fiber knob consists primarily of residues fromthe AB loop and CD loop and extends secondarily to the FG and HI loopand the B, E and F β sheets (Roelvink et al. 1999, Bewley et al. 1999).The HI loop has been the best studied insertion site on the Fiber knob(Worgall et al. 2004, Mizuguchi and Hayakawa 2004, Koizumi et al. 2003,Belousova et al. 2002, Noureddini and Curiel 2005, Nicklin et al. 2001),and incorporation of an epitope into the HI loop (residue 543 and 544)resulted in potent anti-epitope immunity (Krause et al. 2006).Therefore, an immunodominant CS-derived B cell epitope was initiallyinserted into the HI loop of the Fiber protein.

Hexon is the most abundant protein of the adenovirus capsid with 720copies per virion. In the mature virus, Hexon exists as homotrimericcapsomeres which make up the facets of the icosahedral virion (Rux andBurnett 2004). The crystal structures of adenovirus serotypes 2 and 5(Ad2 and Ad5) Hexons have been solved, revealing a complex moleculararchitecture (Athapilly et al. 1994, Roberts et al. 1986, Rux andBurnett 2000). The base of each monomeric subunit consists of twobeta-barrel motifs that are present in the capsid proteins of manyicosahedral viruses. Three long loops (DE1, FG1, and FG2) extend outfrom the base structure to form the tower region of each molecule (Ruxand Burnett 2004). Sequences within these loop domains protrude to thesurface of the capsid to form the exterior of the virion. Alignmentsfrom different adenovirus serotypes show that the sequences located onthe capsid exterior are poorly conserved in both length and amino acidsequence (Crawford-Miksza and Schnurr 1996). Furthermore, it has beenshown that the sequences located in these poorly conserved domains,termed hypervariable regions (HVRs), contain the determinants againstwhich serotype-specific antibodies are produced (Top 1975, Rux andBurnett 2000, Top et al. 1971).

Based on early sequence alignments, seven HVRs were identifiedthroughout the Hexon molecule (Crawford-Miksza and Schnurr 1996, Robertset al 2006). Because the HVRs are poorly conserved between serotypes anddo not appear to be involved in maintaining the structural integrity ofHexon, small changes could be made to these domains without affectingthe viability of the virus (Rux and Burnett 2000). For example ahexahistidine tag can be inserted into HVR2, HVR3, HVR5, HVR6, and HVR7without compromising virus viability (Wu et al. 2005). Thus, Hexon HVRsare often used as targets to efficiently induce an antibody responseagainst peptides located in Hexon HVRs (Worgall et al. 2005, Crompton etal. 1994). Due to its poor conservation in length between serotypes andits position on the outermost surface of the adenovirus capsid (Rux andBurnett 2000, Crawford-Miksza and Schnurr 1996), Hexon HVR5 wasinitially chosen as a site for epitope insertion. Further, the crystalstructure of Hexon indicates that HVR5 is a flexible loop on the capsidsurface, suggesting that HVR5 can accommodate relatively large peptideswithout compromising the structural integrity of the capsid (Roberts etal. 1986). Hexon-specific CD4+ and CD8+ epitopes have recently beenidentified (Leen et al. 2008), and the CD4+ T cell response toadenovirus is focused against conserved residues within the Hexonprotein in humans (Onion et al. 2007, Heemskerk et al. 2006).

The adenovirus core is composed of the viral genome and four coreproteins. The terminal protein (TP) is covalently linked to the 5′ endof each linear viral DNA strand at two copies per virion. Noncovalentlyand nonspecifically bound to the viral DNA through arginine-richportions are three other core proteins mu (μ), V (pV) and VII (pVII).pVII is the major core protein contributing roughly 700-800 copies pervirion, and serves as a histone-like center around which viral DNA iswrapped to form nucleosome structures.

Modification of Adenovirus Capsid Proteins to Enhance Humoral Immunity

In some embodiments, circumsporozoite (CS) adenoviral vectors that havean immunodominant CS protein B epitope in an adenovirus capsid protein(inserted in the Hexon or Fiber) are described. The transgene may beunder a promoter such as CMV to augment cell-mediated and humoral immuneresponses to CS protein.

A central repeat region is the conserved structure of CS protein amongPlasmodium species, and antibody against this repeat sequence has beenshown to have sporozoite neutralizing activity. Examples of a repeatsequence in Plasmodium CS protein are (NANP)_(n) repeat (P. falciparum;SEQ ID NO:60), ANGAGNQPG repeat (P. vivax; SEQ ID NO:63) and NAAG repeat(P. malariae; SEQ ID NO:64), which can be inserted into adenoviruscapsid proteins. In some embodiments, four or more (NANP)_(n) repeats(SEQ ID NO:60) of PfCSP may be inserted into HVR1 of adenovirus serotype5 Hexon. In some embodiments, two, four, six, eight, ten, fourteen,sixteen, eighteen, twenty, twenty-two, twenty-four, twenty-six, ortwenty-eight (NANP)_(n) repeats (SEQ ID NO:60; n=2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28) of PfCSP are inserted into HVR1 ofadenovirus serotype 5 Hexon. The (NANP)_(n) repeat sequence may beadditionally inserted in the in the HI loop of Fiber.

In some embodiments, immunodominant neutralizing B cell epitopes to CSwere mapped to develop improved CS protein adenovirus vaccines. Miceimmunized with recombinant P. yoelii CS protein (PyCS) generated hightiters against the two major immunodominant B epitopes, QGPGAP (SEQ IDNO:59) and QQPP (SEQ ID NO:65), but the in vitro neutralization assayindicated that the humoral immune response to QGPGAP epitope (SEQ IDNO:59) itself may account for neutralizing activity because theneutralization could be reversed by adding (QGPGAP)₃ peptide (SEQ IDNO:59; n=3) to the medium. In some embodiments, three or more(QGPGAP)_(n) repeats (SEQ ID NO:59) of PyCS are inserted into HVR1 ofadenovirus serotype 5 Hexon. In some embodiments, three, four, five,six, seven, eight, nine, ten, eleven, or twelve (QGPGAP)_(n) repeats(SEQ ID NO:59; n=3, 4, 5, 6, 7, 8, 9, 10, 11, 12) of PyCS may beinserted into HVR1 of adenovirus serotype 5 Hexon. The (QGPGAP)_(n)repeat sequence may be additionally inserted in the in the HI loop ofFiber.

The B cell epitope peptide should be presented on the surface ofadenovirus virions so that immune system can recognize the epitopeefficiently. Such insertion sites could be HVRs of Hexon and Loopstructures in Fiber, and different insertion sites can be combined.

Modification of Adenovirus Capsid and Core Proteins to Enhance T HelperCell Activation

In another embodiment, a CD4+ epitope specific to the transgene used inan adenoviral vector may be incorporated into adenovirus proteins suchas pVII, pV and Hexon to augment immunogenicity of the adenoviral-basedvaccine. Professional antigen presenting cells (APC) such as dendriticcells (DC) and B cells can uptake particulated pathogens like virusparticles via endocytosis and present CD4+ epitopes in the pathogen toCD4+ T cells which acts as helper cells for humoral and/or cellularimmune responses. pVII and Hexon may easily be used as adenovirus targetproteins to insert antigenic CD4+ peptides because of high copy numberof pVII (700-800 copies) and Hexon (720 copies) in one virion.

Modification of Adenovirus Capsid Proteins to Circumvent ExistingAdenovirus Immunity

In some embodiments, adenovirus Fiber and Hexon capsid proteins may bemodified to insert a B cell or T helper cell epitope to overcomeexisting immunity to adenovirus and/or enhance the humoral response toan adenovirus vaccine. An estimated 80% of young adults in humanpopulation have circulating neutralizing antibodies to adenovirus(Douglas 2007), especially to serotype 5 (Ad5). In studies utilizingadenovirus as a gene therapy vector, it was found that the presence ofneutralizing antibodies in animals limits the expression of transgenesdelivered by adenovirus. In addition to neutralizing antibodies, CD8+ Tcell responses also contributed to the limitation of recombinant geneexpression (Yang et al. 1995, Yang et al 1996). Such pre-existingimmunity to adenovirus has previously been reported to inhibit theefficacy of a recombinant adenovirus vaccine (Papp et al. 1999) and alsoreduces immunogenicity of adenovirus-based vaccines in a clinical trial(Priddy et al. 2008).

Hexon is a major target for anti-Ad capsid immune responses (Roy et al.2005, Wohlfart 1988), and is likely responsible for the potent adjuvanteffect of adenovirus, including the induction of CD4+ and CD8+ T cellresponses. Therefore, one strategy that has been employed to circumventpre-existing anti-adenovirus immunity is to replace all or part of theHexon with a different protein, for example, rare serotypes such asadenovirus 11, 24, 26 and 35. Because Hexon is a major target ofanti-adenovirus neutralizing antibody (Youil et al. 2002, Sumida et al.2005), the entire Hexon or HVRs of Hexon may be swapped with the rareserotypes (Wu et al. 2002, Roberts et al. 2006).

In another strategy as described in one embodiment herein, an adenoviralHexon may be modified by replacement of HVR1 or HVR5 with an antigenicpeptide to circumvent pre-existing anti-adenovirus immunity oranti-adenovirus neutralizing antibody induced by previous vaccinationwith adenoviral vector. In some embodiments, an antigenic peptide may bean immunogenic epitope of Plasmodium CS protein, and in certain aspects,the epitope may comprise a central repeat sequence, CD4+ epitopesequence or CD8+ epitope sequence.

Repeat administration with an Ad vector of the same serotype isprevented due to anti-Ad immunity following immunization. Therefore,many Ad vaccines impede boosting of the vaccine by preventing expressionand presentation of the antigen encoded by the transgene (Yang 1995,Hackett et al. 2000, Harvey et al. 1999, Mastrangeli et al. 1996). Theaddition of a specific epitope to the Ad capsid, such as those describedin the examples below, may reduce or eliminate this impediment accordingto some embodiments.

The following examples are provided to better illustrate the embodimentsand are not to be interpreted as limiting the scope of any claimedembodiment. The extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLE 1 Construction of Capsid-Modified Plasmodium CircumsporozoiteProtein Adenovirus Plasmid Vectors and Recombinant Adenovirus Particles

Epitope Mapping

First, an immunodominant neutralizing B cell epitope in PyCS was chosen.Naïve Balb/c mice were immunized with recombinant PyCS-protein withincomplete freund adjuvant three times and the pooled serum was used todetermine the critical epitope of neutralizing antibody.

Briefly, in the neutralizing assay, human CD81 expressing HepG2 cellswere used as target cells. CD81 is a molecule necessary for malariaparasites to form parasitophorous vacuoles in hepatocytes where theymultiply and develop into schizonts (Silvie et al 2006), thus greatlyincreasing the in vitro infectivity of sporozoites.

In this assay, PyCS synthetic peptides were added to the wells to blockpeptide specific antibody. The results of epitope mapping indicated thatthe PyCS central repeat sequence, QGPGAP (SEQ ID NO:59), is a morepotent neutralizing epitope in PyCS than QQPP (SEQ ID NO:65). Therefore,insertion of the (QGPGAP)_(n) epitope (SEQ ID NO:59) was used to modifyadenovirus capsid proteins, Hexon and/or Fiber.

Construction of Capsid-Modified Plasmid Vectors

Adenovirus shuttle vector pShuttle-CMV (STRATAGENE) was modified byinserting a GFP-expression cassette under the cloning site. First,BsmBI-SacI fragment (pCMV+GFP) and SacI-BsmBI fragment (SV40 poly Asignal) of pmaxGFP (Lonza, Cologne, Germany) were blunted and insertedinto the blunted SalI and KpnI sites of pUC19 respectively. TheBamHI-EcoRI fragment of the resulting pCMV-GFP/pUC19 was inserted intothe same sites of SV40pA/pUC19 to create SV40pA-pCMV-GFP fragment. Thefragment was blunted and inserted into the EcoRV site of pShuttle-CMV.The resulting shuttle vector (GFP/pShuttle-CMV) has dual pCMV promotersand SV40pAs for a transgene and GFP.

Another modification of the Adenovirus shuttle vector pShuttle-CMV wasdone to replace the CMV promoter region with CMV5 promoter frompQBI-AdCMV5 (QBIOgene). The SgrAI-KpnI fragment of pShuttle-CMV wasreplaced with the fragment containing the CMV5 promoter sequence and theupstream sequence from CMV promoter in pShuttle-CMV to constructpShuttle-CMV5 vector.

The P. yoelii CS (PyCS) gene was codon-optimized except for the(QGPGAP)_(n) repeats (SEQ ID NO:59) by overlapping PCR reaction based onJCat codon-optimization algorithm (http://www jcat de/).

The PfCSP amino acid sequence of P. falciparum 3D7 strain was used as atemplate sequence for codon-optimization. Codon-optimization for proteinexpression in humans was done by Integrated DNA Technologies'(Coralville, Iowa USA) optimization software. DNA fragments that encodewhole PfCSP except for the GPI-anchored motif at the C-terminus (FIG.10; SEQ ID NO:2) were synthesized by Integrated DNA Technologies

Codon-optimized PyCS gene (FIG. 9; SEQ ID NO:1) or PfCSP gene (FIG. 10;SEQ ID NO:2) was inserted into KpnI and HindIII sites of pShuttle-CMV,pShuttle-CMV5, or GFP/pShuttle-CMV. The resulting Plasmodiumcircumsporozoite protein coding adenovirus shuttle vectors were used forhomologous recombination with AdEasy-1 to construct adenovirus genomewhich has Plasmodium circumsporozoite antigenic gene and intactadenovirus protein coding sequences. Briefly, Plasmodiumcircumsporozoite protein coding adenovirus shuttle vectors werelinearized by PmeI digestion, and E. coli BJ5183 cells wereco-transformed with the linearized shuttle vector and pAdEasy-1 vector(Bruna-Romero et al 2003) for homologous recombination.

Modification of adenovirus capsid proteins is summarized and illustratedin FIG. 1. Modification of HVR1 sequence in the adenovirus genome DNA isillustrated in FIG. 2. Briefly, AdEasy-1 was digested with SfiI and the6.4 kbp fragment was subcloned into EcoRI and PstI sites of pUC19 usingEcoRI-SfiI and PstI-SfiI linker oligomers. To replace HVR1 with aPlasmodium circumsporozoite protein B cell epitope, the regioncontaining AgeI and NdeI sites was amplified by two-step PCR usingprimers which have the epitope sequence instead of HVR1 sequence. ThePCR product was digested with AgeI and NdeI, and then used to replacethe native AgeI-NdeI region of SfiI fragment in SfiI/pUC19 vector. Afterconfirming the sequence, the SfiI fragment of adenovirus genome DNA wasreplaced with the SfiI fragment containing the circumsporozoite epitopesequence to produce an HVR1-modified Hexon. In some embodiments, anHVR-modified Hexon may have a nucleic acid sequence of SEQ ID NO:3 (FIG.11), SEQ ID NO:4 (FIG. 12), SEQ ID NO:5 (FIG. 13), SEQ ID NO:6 (FIG.14), SEQ ID NO:7 (FIG. 15), SEQ ID NO:8 (FIG. 16), SEQ ID NO:9 (FIG.17), SEQ ID NO:10 (FIG. 18), SEQ ID NO:11 (FIG. 19), SEQ ID NO:12 (FIG.20), SEQ ID NO:13 (FIG. 21), SEQ ID NO:14 (FIG. 22), SEQ ID NO:15 (FIG.23), SEQ ID NO:16 (FIG. 24), SEQ ID NO:17 (FIG. 25), SEQ ID NO:18 (FIG.26), SEQ ID NO:19 (FIG. 27), SEQ ID NO:20 (FIG. 28), SEQ ID NO:21 (FIG.29), SEQ ID NO:22 (FIG. 30), or SEQ ID NO:23 (FIG. 31).

To insert (NANP)₂₈ (SEQ ID NO:60; n=28) in HVR1, a part of the centralrepeat region of codon-optimized PfCSP was amplified by PCR usingprimers having hexon-specific sequence at 5′ and NANP-specific sequenceat 3′, and the resulting DNA fragment was inserted into the AgeI-NdeIregion by second PCR.

For HVR5-modification, as illustrated in FIG. 3, XbaI site wasintroduced into HVR5 in the L1 Loop of Hexon in AdEasy-1 and thensynthesized, phosphorylated double strand oligomer coding the Plasmodiumcircumsporozoite protein epitope was inserted into the XbaI site. Theinsertion was confirmed by sequencing (FIG. 31; SEQ ID NO:23).

For Fiber-modification, as illustrated in FIG. 4, the SpeI-PacI fragmentof AdEasy-1 was subcloned into EcoRI and PstI sites of pUC19 usingEcoRI-Pact and PstI-SpeI linker oligomers. To insert a Plasmodiumcircumsporozoite protein B-cell epitope sequence into HI loop of Fiberknob, the region containing EcoNI (or NheI) and MfeI sites was amplifiedby two-step PCR using primers which have the epitope sequence. The PCRproduct was digested with EcoNI (or NheI) and MfeI, and then used toreplace the native EcoNI (or NheI)-MfeI region of Fiber inSpeI-PacI/pUC19 vector. After confirming the sequence (FIG. 32, SEQ IDNO:24; FIG. 33, SEQ ID NO:25), the SpeI-Pact fragment of AdEasy-1 wasreplaced with the SpeI-Pact fragment containing the epitope sequence.The resulting Fiber-modified adenovirus DNA was used for homologousrecombination with Plasmodium circumsporozoite protein coding adenovirusshuttle vector to produce Fiber-modified Plasmodium circumsporozoiteprotein adenovirus DNA.

To construct HVR1 and Fiber-modified adenovirus DNA which has twoepitope insertions, SfiI-SfiI fragment of Fiber-modified adenovirus DNAwas replaced with SfiI-SfiI fragment having the circumsporozoite proteinepitope in HVR1 as illustrated in FIG. 5.

To modify the C-terminus of pVII, the region containing Sfi I and Sal Isites was amplified by two-step PCR using primers which have thecircumsporozoite protein epitope sequence. The PCR product was digestedwith SfiI and SalI, and then used to replace the native SfiI-SalI regionof SfiI/pUC19 vector (FIGS. 6, 7 and 8). After confirming the sequence(FIG. 34, SEQ ID NO:26; FIG. 35, SEQ ID NO:27), the SfiI-SfiI fragmentof HVR1 and/or Fiber-modified circumsporozoite protein adenovirus DNAwas replaced with SfiI-SfiI fragment having the circumsporozoite proteinepitope in pVII.

To insert the circumsporozoite protein CD4+ epitope sequenceEYLNKIQNSLSTEWSPCSVT (SEQ ID NO:62) in the middle of pVII, about 7.7 kbfragment of pAdEasy-1 was prepared by RsrII digestion and cloned betweenthe EcoRI and HindIII sites of pUC19 plasmid using RsrII linker(RsrII/pUC19). The region containing AscI and BglII sites in RsrII/pUC19was amplified by two-step PCR using primers which have the epitopesequence. The PCR product was digested with AscI and BglII, and thenused to replace the native AscI and BglII region in RsrII/pUC19 plasmid.After confirming the sequence of the replaced region (FIG. 36, SEQ IDNO:28; FIG. 37, SEQ ID NO:29), the RsrII fragment of HVR1-modifiedadenovirus DNA was replaced with the RsrII fragment containing theepitope sequence.

The recombinant adenoviruses listed in Table 1 (P. yoelii) and Table 2(P. falciparum) below were produced to evaluate the effect of epitopeinsertion on infectivity, immunogenicity and sensitivity topre-existing, anti-adenovirus immunity. Recombinant adenovirus vectorsused were replication defective, E1 and E3-deleted adenovirus serotype 5(STRATAGENE). FIG. 1 shows the schematic structure of capsid-modifiedPlasmodium circumsporozoite protein recombinant adenovirus.

TABLE 1 Recombinant adenoviruses (Plasmodium yoelii circumsporozoiteprotein) Adeno- Recombinant Pro- Trans- virus Insertion AntigenAdenovirus moter gene Protein Site Position (a.a.) Inserted SequenceLength P. yoelii wt/Empty CMV None Hexon — — — — circum- Fiber — — — —sporozoite pVII — — — — protein wt/GFP CMV GFP Hexon — — — — (PyCS)Fiber — — — — pVII — — — — wt/PyCS-GFP CMV PyCS + Hexon — — — — GFPFiber — — — — pVII — — — — (QGPGAP)₃- CMV PyCS + Hexon HVR1 from 138 to164 (QGPGAP)₃ 18 HVR1/PyCS-GFP GFP Fiber — — — — pVII — — — — (QGPGAP)₃-CMV PyCS + Hexon HVR5 between 268 and 269 (QGPGAP)₃ 18 HVR5/PyCS-GFP GFPFiber — — — — pVII — — — — (QGPGAP)₃- CMV PyCS + Hexon — — — —Fib/PyCS-GFP GFP Fiber HI Loop between 543 and 544 (QGPGAP)₃ 18 pVII — —— — (QGPGAP)₃- CMV PyCS + Hexon HVR1 from 138 to 164 (QGPGAP)₃ 18HVR1/Fib/PyCS- GFP Fiber HI Loop between 543 and 544 (QGPGAP)₃ 18 GFPpVII — — — — (QGPGAP)₃- CMV PyCS + Hexon — — — — Fib/PyCD4-pVII-1/ GFPFiber HI Loop between 543 and 544 (QGPGAP)₃ 18 PyCS-GFP pVII C-terminusbetween 198 and YNRNIVNRLLGDALNGKPEEK 21 STOP Codon wt/cmv5-PyCS CMV5PyCS Hexon — — — — Fiber — — — — pVII — — — — (QGPGAP)₃- CMV5 PyCS HexonHVR1 from 138 to 164 (QGPGAP)₃ 18 HVR1/cmv5-PyCS Fiber — — — — pVII — —— — (QGPGAP)₄-HVR1/ CMV5 PyCS Hexon HVR1 from 138 to 164 (QGPGAP)₄ 24cmv5-PyCS Fiber — — — — pVII — — — — (QGPGAP)₅-HVR1/ CMV5 PyCS HexonHVR1 from 138 to 164 (QGPGAP)₅ 30 cmv5-PyCS Fiber — — — — pVII — — — —(QGPGAP)₆- CMV5 PyCS Hexon HVR1 from 138 to 164 (QGPGAP)₆ 36HVR1/cmv5-PyCS Fiber — — — — pVII — — — — (QGPGAP)₇- CMV5 PyCS HexonHVR1 from 138 to 164 (QGPGAP)₇ 42 HVR1/cmv5-PyCS Fiber — — — — pVII — —— — (QGPGAP)₈- CMV5 PyCS Hexon HVR1 from 138 to 164 (QGPGAP)₈ 48HVR1/cmv5-PyCS Fiber — — — — pVII — — — — (QGPGAP)₉- CMV5 PyCS HexonHVR1 from 138 to 164 (QGPGAP)₉ 54 HVR1/cmv5-PyCS Fiber — — — — pVII — —— — (QGPGAP)₁₁- CMV5 PyCS Hexon HVR1 from 138 to 164 (QGPGAP)₁₁ 66HVR1/cmv5-PyCS Fiber — — — — pVII — — — — (QGPGAP)₁₂- CMV5 PyCS HexonHVR1 from 138 to 164 (QGPGAP)₁₂ 72 HVR1/cmv5-PyCS Fiber — — — — pVII — —— —

TABLE 2 Recombinant adenoviruses (Plasmodium falciparum circumsporozoiteprotein) Adeno- Recombinant Pro- Trans- virus Insertion AntigenAdenovirus moter gene Protein Site Position (a.a.) Inserted SequenceLength P. falciparum wt/PfCSP CMV PfCSP Hexon — — — — circum- Fiber — —— — sporozoite pVII — — — — protein (NANP)₄- CMV PfCSP Hexon HVR1 from138 to 164 (NANP)₄ 16 (PfCSP) HVR1/PfCSP Fiber — — — — pVII — — — —(NANP)₄- CMV PfCSP Hexon — — — — Fib/PfCSP Fiber HI Loop between 543 and544 (NANP)₄ 16 pVII — — — — (NANP)₄- CMV PfCSP Hexon HVR1 from 138 to164 (NANP)₄ 16 HVR1/Fib/PfCSP Fiber HI Loop between 543 and 544 (NANP)₄16 pVII — — — — (NANP)₆- CMV PfCSP Hexon HVR1 from 138 to 164 (NANP)₆ 24HVR1/PfCSP Fiber — — — — pVII — — — — (NANP)₈- CMV PfCSP Hexon HVR1 from138 to 164 (NANP)₈ 32 HVR1/PfCSP Fiber — — — — pVII — — — — (NANP)₁₀-CMV PfCSP Hexon HVR1 from 138 to 164 (NANP)₁₀ 40 HVR1/PfCSP Fiber — — —— pVII — — — — (NANP)₁₂- CMV PfCSP Hexon HVR1 from 138 to 164 (NANP)₁₂48 HVR1/PfCSP Fiber — — — — pVII — — — — (NANP)₁₄- CMV PfCSP Hexon HVR1from 138 to 164 (NANP)₁₄ 56 HVR1/PfCSP Fiber — — — — pVII — — — —(NANP)₁₆- CMV PfCSP Hexon HVR1 from 138 to 164 (NANP)₁₆ 64 HVR1/PfCSPFiber — — — — pVII — — — — (NANP)₁₈- CMV PfCSP Hexon HVR1 from 138 to164 (NANP)₁₈ 72 HVR1/PfCSP Fiber — — — — pVII — — — — (NANP)₂₀- CMVPfCSP Hexon HVR1 from 138 to 164 (NANP)₂₀ 80 HVR1/PfCSP Fiber — — — —pVII — — — — (NANP)₂₂- CMV PfCSP Hexon HVR1 from 138 to 164 (NANP)₂₂ 88HVR1/PfCSP Fiber — — — — pVII — — — — wt/cmv5-PfCSP CMV5 PfCSP Hexon — —— — Fiber — — — — pVII — — — — (NANP)₂₂- CMV5 PfCSP Hexon HVR1 from 138to 164 (NANP)₂₂ 88 HVR1/cmv5- Fiber — — — — PfCSP pVII — — — — (NANP)₂₈-CMV5 PfCSP Hexon HVR1 from 138 to 164 (NANP)₁₇(NVDP)₁(NANP)₁₀ 112 HVR1/cmv5- Fiber — — — — PfCSP pVII — — — — (NANP)₄- CMV PfCSP HexonHVR1 from 138 to 164 (NANP)₄ 16 HVR1/PfCD4- Fiber — — — — pVII-1/PfCSPpVII C-terminus between 198 and EYLNKIQNSLSTEWSPCSVT 20 STOP Codon(NANP)₄- CMV PfCSP Hexon — — — — Fib/PfCD4-pVII- Fiber HI Loop between543 and 544 (NANP)₄ 16 1/PfCSP pVII C-terminus between 198 andEYLNKIQNSLSTEWSPCSVT 20 STOP Codon (NANP)₄- CMV PfCSP Hexon HVR1 from138 to 164 (NANP)₄ 16 HVR1/Fib/PfCD4- Fiber — — — — pVII-1/PfCSP pVIIC-terminus between 198 and EYLNKIQNSLSTEWSPCSVT 20 STOP Codon (NANP)₄-CMV PfCSP Hexon HVR1 from 138 to 164 (NANP)₄ 16 HVR1/PfCD4- Fiber — — —— pVII-2/PfCSP pVII Middle between 92 and EYLNKIQNSLSTEWSPCSVT 20 93Codon (NANP)₄- CMV PfCSP Hexon HVR1 from 138 to 164 (NANP)₄ 16HVR1/PfCD4- Fiber — — — — pVII-3/PfCSP pVII Middle between 140 andEYLNKIQNSLSTEWSPCSVT 20 141 Codon (NANP)₂₂- CMV5 PfCSP Hexon HVR1 from138 to 164 (NANP)₂₂ 88 HVR1/PfCD4- Fiber — — — — pVII-3/cmv5- pVIIMiddle between 140 and EYLNKIQNSLSTEWSPCSVT 20 PfCSP 141 Codon (NANP)₂₈-CMV5 PfCSP Hexon HVR1 from 138 to 164 (NANP)₁₇(NVDP)₁(NANP)₁₀ 112 HVR1/PfCD4- Fiber — — — — pVII-3/cmv5- pVII Middle between 140 andEYLNKIQNSLSTEWSPCSVT 20 PfCSP 141 Codon

The capsid-modified adenovirus genome DNA plasmid was purified,linearized by PacI digestion, and used for transfection of AD293 cells.

Adenovirus particles were prepared from the transfected AD293 cells byfour rounds of freeze/thaw and used for further virus amplification.After the last amplification, adenovirus particles were purified by CsClgradient centrifugation. The band was then collected and dialyzedagainst dialysis buffer to remove CsCl. Virus particle (v.p.) wascalculated based on O.D. 260 (1 O.D.260=1.25×10¹² v.p./mL) (Bruna-Romeroet al. 2003).

During the adenovirus amplification procedure, small differences inadenovirus growth were observed among capsid-modified adenoviruses,demonstrating that adenovirus infectivity and productivity was notadversely affected by the modification.

EXAMPLE 2 Plasmodium yoelli Circumsporozoite Protein-Specific ImmuneResponse

Validation of Plasmodium yoelii Recombinant Adenoviruses

Plasmodium circumsporozoite protein coding adenovirus shuttle vectorswere used for transient transfection to confirm Plasmodiumcircumsporozoite protein expression using AD293 cells (FIG. 38). 24hours after transfection, cells were lysed in SDS sample buffer followedby SDS PAGE electrophoresis and western blotting with anti-PyCSmonoclonal antibody (9D3).

To confirm the epitope insertion into adenovirus capsid proteins,purified recombinant adenoviruses were analyzed by SDS-PAGE (2×10⁹v.p./lane) and Western blot (1×10⁹ v.p./lane) with anti-sporozoiteantibody which recognizes the (QGPGAP)_(n) (SEQ ID NO:59) repeats weredone as shown in FIGS. 39A and 40A. The intensity of the bands in FIG.39A correlated with the copy number of the capsid protein in anadenovirus virion: the copy number of Fiber (36 copies per virion) istwenty-times less than Hexon (720 copies per virion). The lower band inlane 4 in FIG. 39A is likely a degraded Hexon. The intensity of thebands in FIG. 40A correlated with the number of (QGPGAP)_(n) (SEQ IDNO:59) repeats inserted into HVR1.

To assess whether the PyCS-B epitope was exposed to the outside ofadenovirus virion, serially diluted purified recombinant adenovirusparticles were coated onto Enzyme-Linked Immunosorbent Assay (ELISA)plate and detected with anti-PyCS antibody that recognizes (QGPGAP)_(n)(SEQ ID NO:59) repeats. The antibody recognized all the capsid-modifiedadenoviruses (FIGS. 39B and 40B). The results of ELISA assay suggestthat the PyCS-B epitope incorporated in capsid proteins were wellexposed to the outside of adenovirus virions.

Plasmodium Yoelii Circumsporozoite Protein-Specific Immune Responseafter Immunization with Capsid-Modified PyCS Adenovirus

Six- to eight-week old female BALB/c mice were purchased from Taconic(Hudson, N.Y., USA) and maintained under standard conditions in theLaboratory Animal Research Center of The Rockefeller University. Forimmunization, adenoviruses were diluted in PBS and injectedintramuscularly at indicated doses.

To evaluate the immunogenicity of recombinant adenoviruses after asingle immunization, groups of naïve BALB/c mice (five per group) wereimmunized with 1×10⁹v.p. of various recombinant PyCS adenovirusesintramuscularly and PyCS-specific cell-mediated immune responses (CMI)were measured by ELISPOT 2 weeks after immunization (FIG. 41A).

The number of PyCS-specific, IFN-γ-secreting CD8+ T cells in the spleensof immunized mice were determined by an ELISPOT assay, using a syntheticpeptide corresponding to the CD8+ T cell epitope (SYVPSAEQI; SEQ IDNO:66) within the PyCS protein. Briefly, 96 well nitrocellulose plates(Milititer HA, Millipore) were coated overnight with anti-mouseinterferon γ mAb, R4. After overnight incubation at room temperature,the wells were washed repeatedly with culture medium and blocked withculture medium for 4 hours. 5×10⁵ Splenocytes from immunized mice wereadded to the ELISPOT wells in the presence or absence of 10 μg/mL CD8+ Tcell epitope peptide and incubated 24 hours at 37° C. and 5% CO₂. Afterextensive washing of the plates with PBS containing 0.05% Tween 20(PBST), biotinylated anti-mouse interferon γ mAb, XMG1.2, in PBST wereadded and incubated overnight at 40° C. After washing with PBST, theplates will be incubated with peroxidase-labeled avidin (eBiosciences).The spots were developed by adding AEC substrate (BD Biosciences).

All of the capsid-modified adenoviruses induced comparable level of CMIto adenoviruses having intact capsid protein at this dose (FIG. 41B).

Next, naïve BALB/c mice were given multiple doses of recombinantadenoviruses with increasing doses, i.e. 1×10⁸, 1×10⁹, and 1×10¹⁰ v.p.,at 3 week intervals, as shown in FIG. 42A. PyCS-specific humoralresponse was determined by ELISA. Five microliters of blood wascollected from tail vein of the immunized mice and diluted in 495 μl ofPBS, and then the samples were centrifuged at 5,000 rpm for 5 min toprepare diluted plasma samples (×100). Maxisorp ELISA plates were coatedwith 5 μg/ml CS-specific peptide ((QGPGAP)₃; SEQ ID NO:59, n=3) in 0.1 MSodium Carbonate Buffer (pH 9.5) at 4° C. for overnight. Plates werewashed and blocked with 1× Diluent for 2 hours at room temperature. Theplates were washed again and 100 μl of serially twofold-diluted plasmaor serum in 1× Diluent was added to the plates and the plates wereincubated for one hour at room temperature. The plates were washed andincubated with 100 μl of HRP-labeled goat anti-mouse IgG antibody. Allof the peptides were synthesized by Biosyntheis (Lewisville, Tex., USA).

Using this immunization regimen, all capsid-modified adenovirusesinduced a significantly higher level of anti-(QGPGAP)₃ antibody responsethan wt/PyCS-GFP at week 10 (FIG. 42B).

To determine the vaccine efficacy of capsid-modified adenovirus, theimmunized mice were challenged with 2×10⁴ infectious P. yoeliisporozoites via tail vein injection at week 10. Parasite burden 42 hoursafter sporozoite challenge was determined by quantifying the amounts ofparasite-specific ribosomal RNA in mouse liver and described as a ratioof the absolute copy number of parasite ribosomal RNA to that of mouseGAPDH mRNA. For statistical analysis, the values were log-transformedand then one-way ANOVA followed by a Dunnett's test was employed todetermine the differences.

Vaccinations with (QGPGAP)₃—HVR1/PyCS-GFP, (QGPGAP)₃-Fib/PyCS-GFP or(QGPGAP)₃—HVR1/Fib/PyCS-GFP induced a higher level of protection thanwt/PyCS-GFP, resulting in a significantly lower parasite burden in themalaria challenged mice (FIG. 42C).

Next, the functionality of PyCS-specific antibody induced bycapsid-modified adenoviruses was evaluated. First, to test whether thesera of adenovirus-immunized mice at week 10 (FIG. 42A) could recognizeintact sporozoites, an indirect immunofluorescene assay (IFA) wasperformed. In IFA, air-dried sporozoites on multi-spot glass slides wereincubated with 3% Bovine Serum albumin (BSA) in PBS for one hour andthen incubated with diluted sera for one hour. After washing, the slideswere incubated with fluorescent-labeled secondary antibody for one hour.The slides were washed and IFA titers were determined as the highestdilution producing fluorescence under a fluorescent microscope. Both(QGPGAP)₃-HVR1/PyCS-GFP and (QGPGAP)₃-HVR1/Fib/PyCS-GFP induced ahighest IFA titer against sporozoites (FIG. 43A), indicating that theinsertion of (QGPGAP)₃ epitope in HVR1 of adenovirus Hexon enabled PyCSadenovirus to elicit a robust antibody response against not only asynthetic peptide, but also a native epitope present in the malariaparasites.

Second, to determine whether mice immunized with capsid-modifiedadenovirus (FIG. 42A) developed “functional” antibodies that couldneutralize the infectivity of sporozoites, an in vitro sporozoiteneutralization assay was performed.

In the in vitro neutralizing assay, P. yoelii sporozoites were added toCD81/HepG2 in a 96-well plate in the presence of 30-fold diluted pooledserum from adenovirus-immunized mice. After a two-hour incubation,uninfected sporozoites were washed out with medium and then the cellswere cultured for 42 hours. Relative amount of parasite ribosomal RNA tohuman GAPDH mRNA was measured by real-time PCR (Ophorst et al. 2006).

The pooled serum samples from mice immunized with capsid-modifiedadenovirus, particularly (QGPGAP)₃-HVR1/PyCS-GFP and(QGPGAP)₃-HVR1/Fib/PyCS-GFP, almost completely inhibited (99%) thesporozoite infectivity in vitro (FIG. 43B). It is noted that the degreeof inhibition in this assay was inversely correlated with the IFA titersshown in FIG. 43A.

Protection from Blood Stage Malaria Infection

Next it was determined whether immunization with capsid-modified rAdprotects mice from developing a blood-stage malaria infection aftersporozoite challenge. The experiments were performed twice and in eachexperiment, 20 BALB/c mice in each group were immunized three times withwt/PyCS-GFP or (QGPGAP)₃-HVR1/PyCS-GFP as shown in FIG. 42A and at 4weeks after the last immunization, the mice were intravenouslychallenged with 50 P. yoelli sporozoites. Giemsa-stained blood smearswere analyzed from 3 to 12 days after challenge to detect blood stagemalaria parasite infection. In the wt/CS-GFP immunized group, 30 out of40 mice (75%) were infected whereas 35 out of 40 (87.5%) became infectedin the naïve group (Table 3, below). (QGPGAP)₃—HVR1/CS-GFP immunizedmice were more protected than wt/CS-GFP; only 15 out of 40 (37.5%) ofwhich became infected, which is consistent with the result of protectionexperiment measured by parasite burden in liver (FIG. 42C).

TABLE 3 Detection of blood stage malaria parasite infection afterimmunization with wt/PyCS-GFP or (QGPGAP)₃-HVR1/PyCS-GFP. No. of MiceNo. of Mice Protection Immunization (Chllenged) (Infected) (%)Experiment 1 None 20 18 10 wt/PyCS-GFP 20 14 30 (QGPGAP)₃-HVR1/PyCS-GFP20 10 50 Experiment 2 None 20 17 15 wt/PyCS-GFP 20 16 20(QGPGAP)₃-HVR1/PyCS-GFP 20 5 75 Total None 40 35 87.5 wt/PyCS-GFP 40 3075 (QGPGAP)₃-HVR1/PyCS-GFP 40 15 37.5

Prime-Boost Immunization 1

Next, naïve BALB/c mice were given “boosts” of HVR1-modified PyCSadenoviruses which have four or six repeats of (QGPGAP)_(n) (SEQ IDNO:59; n=4, 6) with or without the adjuvant at multiple increasing doses(i.e. 1×10⁸, 1×10⁹, and 1×10¹⁰ v.p.) at 3 week intervals, as shown inFIG. 44A. The adjuvant used in this experiment is Sigma Adjuvant System(Sigma-Aldrich) containing 200 μg/mL Saponin (Sigma-Aldrich). A vial ofSigma Adjuvant System (1 mL) contains 0.5 mg Monophosphoryl Lipid A(detoxified endotoxin) from Salmonella minnesota and 0.5 mg syntheticTrehalose Dicorynomycolate in 2% oil (squalene)-Tween 80 in water.Adenovirus solution was mixed with the equal amount of the adjuvantbefore the immunization. One hundred microliters of theadenovirus-adjuvant mixture was injected intramuscularly. PyCS-specifichumoral and cell-mediated immune responses were measured as describedabove. A trend was observed that HVR1-modified adenovirus having sixrepeats induced higher antibody titer than that having four repeats andthe use of adjuvant augmented the antibody titer (FIG. 44B). Incontrast, there was no effect of the adjuvant on CMI (data not shown).

To determine the vaccine efficacy of HVR1-modified PyCS adenovirus, fivemice in each group were challenged with 2×10⁴ infectious P. yoeliisporozoites via tail vein injection at week 9. Parasite burden 42 hoursafter sporozoite challenge was determined as described above. Forstatistical analysis, the values were log-transformed and then one-wayANOVA followed by a Dunnett's test was employed to determine thedifferences. There was a trend that HVR1-modified adenovirus having sixrepeats reduced parasite burden more than that having four repeats andthe use of adjuvant augmented the protection (FIG. 44C).

To evaluate the functionality of antibody induced by HVR1-modified PyCSadenoviruses, we performed an in vitro sporozoite neutralization assayas described above. Pooled serum samples from mice immunized withHVR1-modified PyCS adenoviruses at week 9 neutralized sporozoiteinvasion at 50-fold dilution (FIG. 44D).

Prime-Boost Immunization 2

Naïve BALB/c mice were given “boosts’ of HVR1-modified PyCS adenoviruseswhich have six, nine, or twelve repeats of (QGPGAP)_(n) (SEQ ID NO: 59;n=6, 9, 12) with or without the adjuvant at three doses of 1×10¹⁰ v.p.at 3 week intervals, as shown in FIG. 45A. The adjuvant used in thisexperiment is Sigma Adjuvant System (Sigma-Aldrich) containing 200 μg/mLSaponin (Sigma-Aldrich). Adenovirus solution was mixed with the equalamount of the adjuvant before the immunization. PyCS-specific humoraland cell-mediated immune responses were measured as described above.HVR1-modified PyCS adenovirus which has twelve repeats of (QGPGAP)_(n)(SEQ ID NO:59; n=12) with the adjuvant induced the highest antibodytiter among the groups at week 9 (FIG. 45B). With respect toPyCS-specific CMI, there was no difference among the groups, indicatingthat the longer epitope insertion up to twelve does not impairadenovirus infectivity in vivo (FIG. 45C). Further, the adjuvant did notaffect the ability of adenovirus to induce CMI (FIG. 45C).

To determine the vaccine efficacy of HVR1-modified PyCS adenovirus, fivemice in each group were challenged with 2×10⁴ infectious P. yoeliisporozoites via tail vein injection at week 9. Parasite burden 42 hoursafter sporozoite challenge was determined as described above. All of theHVR-1 modified PyCS adenovirus having (QGPGAP)_(n) repeats (SEQ IDNO:59, n=6, 9, 12), with or without adjuvant, showed increasedprotection. However, HVR1-modified PyCS adenovirus having twelve repeatsof (QGPGAP)_(n) (SEQ ID NO:59, n=12) with the adjuvant showed the bestprotection (FIG. 45D), which was significantly more protective that anyother treatment.

EXAMPLE 3 Plasmodium falciparum Circumsporozoite Protein-Specific ImmuneResponse

Validation of Plasmodium falciparum Recombinant Adenoviruses

Plasmodium circumsporozoite protein coding adenovirus shuttle vectorswere used for transient transfection to confirm Plasmodiumcircumsporozoite protein expression using AD293 cells (FIG. 46A). 24hours after transfection, cells were lysed in SDS sample buffer followedby SDS PAGE electrophoresis and western blotting with anti-NANPmonoclonal antibody (2A10).

To confirm the epitope insertion into adenovirus capsid proteins,purified recombinant adenoviruses were analyzed by SDS-PAGE (2×10⁹v.p./lane) and Western blot (1×10⁹ v.p./lane) with anti-sporozoiteantibody which recognizes the (NANP)_(n) (SEQ ID NO:60) repeats weredone as shown in FIGS. 47A and 48A. The intensity of the bands in FIG.47A correlated with the copy number of the capsid protein in anadenovirus virion: the copy number of Fiber (36 copies per virion) istwenty-times less than Hexon (720 copies per virion). The intensity ofthe bands in FIG. 48A correlated with the number of NANP repeat HVR1.

To assess whether the PfCSP-B epitope was exposed to the outside ofadenovirus virion, serially diluted purified recombinant adenovirusparticles were coated onto Enzyme-Linked Immunosorbent Assay (ELISA)plate and detected with anti-PyCS antibody that recognizes (NANP)_(n)(SEQ ID NO:60) repeats. The antibody recognized all the capsid-modifiedadenoviruses (FIGS. 47B and 48B). The results of ELISA assay suggestthat the PfCSP-B epitope incorporated in capsid proteins were wellexposed to the outside of adenovirus virions.

Prime-Boost Immunization 3

Naïve BALB/c mice were given multiple, increasing doses of recombinantPfCSP adenoviruses (i.e., 1×10⁸, 1×10⁹, and 1×10¹⁰v.p.) at 3 weekintervals as shown in FIG. 49A. PfCSP-specific humoral response wasdetermined by ELISA as described above using ELISA plates coated with 1μg/ml (T1B)₄, a CS repeat peptide which contains a (NANP)_(n) repeatsequence (SEQ ID NO:60) (Calvo-Calle et al 2006). For statisticalanalysis, the values were log-transformed and one-way ANOVA followed bya Dunnett's test was employed to determine the differences betweenwt/PfCSP and capsid-modified adenoviruses. All capsid-modifiedadenoviruses induced statistically higher anti-NANP antibody titer thanwt/PfCSP.

Prime-Boost Immunization 4

Next, naïve BALB/c mice were given “boosts’ of HVR1-modified PfCSPadenoviruses which have four, six, eight, or ten repeats of (NANP)_(n)(SEQ ID NO:60; n=4, 6, 8, 10) with at multiple increasing doses (i.e.,1×10⁸, 1×10⁹, and 1×10¹⁰ v.p.) at 3 week intervals, as shown in FIG.50A. PfCSP-specific humoral immune responses were measured as describedabove. All of the HVR1-modified adenoviruses induced significantlyhigher anti-NANP antibody titer than wt/PfCSP at week 9 (FIG. 50B). Forstatistical analysis, the values were log-transformed and then one-wayANOVA followed by a Dunnett's test was employed to determine thedifferences.

Prime-Boost Immunization 5

Naïve BALB/c mice were given “boosts” of HVR1-modified adenoviruseswhich have ten, sixteen, or twenty-two repeats of (NANP)_(n) (SEQ IDNO:60; n=10, 16, 22) with or without the adjuvant at three doses of1×10¹⁰ v.p. at 3 week intervals, as shown in FIG. 51A. The adjuvant usedin this experiment is Sigma Adjuvant System (Sigma-Aldrich) containing200 μg/mL Saponin (Sigma-Aldrich). Adenovirus solution was mixed withthe equal amount of the adjuvant before the immunization. PfCSP-specifichumoral immune response was measured as described above, and it wasdetermined that HVR1-modified adenoviruses with longer B cell epitopeinduced higher antibody titer (FIG. 51B).

EXAMPLE 4 PVCS CD4 Epitope Insertion into Adenovirus Core Protein pVII

Antigen-specific CD4 T cells are required for antigen-specific B celldevelopment and proliferation. Therefore to determine whether it wouldbe possible to enhance PyCS-specific humoral immune response induced bycapsid-modified adenovirus by inserting PyCS CD4 epitope in adenovirusprotein, (QGPGAP)₃-Fib/PyCS-GFP that has PyCS CD4 epitope in pVII((QGPGAP)₃-Fib/CD4-pVII-1/PyCS-GFP) was constructed. pVII is one of theadenovirus core proteins and the copy number per virion is 700-800,which is ideal for efficient CD4 epitope presentation onto MHC class IImolecule. As shown in FIG. 52A, the pVII band is shifted by PyCS CD4epitope insertion into pVII on a SDS-PAGE gel.

To test the effect of PyCS CD4 epitope insertion into pVII, naïve BALB/cmice were immunized with (QGPGAP)₃-Fib/PyCS-GFP or(QGPGAP)₃-Fib/CD4-pVII-1/PyCS-GFP as shown in FIG. 42A and anti-QGPGAPantibody titer was determined by ELISA at week 10.(QGPGAP)₃-Fib/CD4-pVII-1/PyCS-GFP induced significantly higheranti-QGPGAP antibody titer than (QGPGAP)₃-Fib/PyCS-GFP (FIG. 52B), andthat indicated PyCS CD4 epitope insertion into pVII augmented humoralimmune response induced by capsid-modified adenovirus.

PfCSP CD4 Epitope Insertion into Adenovirus Core Protein pVII

To evaluate the effect of PfCSP CD4+ epitope insertion into differentpositions in adenovirus core protein pVII on adenovirus-induced immuneresponse, HVR1-modified PfCSP adenoviruses having the PfCSP CD4+ epitopejust before the first Nuclear localization Signal (NLS) or between thetwo NLSs were constructed (FIG. 53A).

To confirm the epitope insertion into pVII, purified recombinantadenoviruses were analyzed by SDS-PAGE as described above. As shown inFIG. 53B, the pVII bands of (NANP)₄—HVR1/CD4-pVII-2/PfCSP and(NANP)₄—HVR1/CD4-pVII-3/PfCSP were shifted upward because of the epitopeinsertion.

PfCSP-Specific Immune Response Induced by HVR1 and pVII-Modified PfCSPAdenovirus

Next, naïve BALB/c mice were given “boosts’ of HVR1 and pVII-modifiedPfCSP adenoviruses which have four repeats of NANP in HVR1 and thePfCD4+ epitope in pVII with at multiple increasing doses (i.e., 1×10⁸,1×10⁹, and 1×10¹⁰v.p.) at 3 week intervals, as shown in FIG. 54A.PfCSP-specific humoral immune responses were measured as describedabove. (NANP)₄—HVR1/CD4-pVII-2/PfCSP and (NANP)₄—HVR1/CD4-pVII-3/PfCSPinduced significantly higher anti-NANP antibody titer than(NANP)₄—HVR1/PfCSP at week 6 (FIG. 54B). In terms of CMI,(NANP)₄—HVR1/CD4-pVII-3/PfCSP induced significantly higher IFNγ andIL-4-secreting PfCSP-specific CD4+ T cells than (NANP)₄—HVR1/PfCSP (FIG.54C).

EXAMPLE 5 Effect of Capsid-Modification on Anti-Adenovirus Immunity

For the in vitro adenovirus neutralization experiments, serum was addedto the AD293 cells at the indicated dilutions prior to the adenovirusinfection. Caucasian serum samples were obtained from InnovativeResearch (Novi, Mich., USA). All flow cytometry data was analyzed withFlowJo v8.8 software (Tree Star, Inc, Ashland, Oreg., USA). AD293 cellswere infected with each capsid-modified adenovirus in the presence ofhuman adenovirus neutralizing serum samples at the indicated dilutionfollowed by measuring GFP expression by flow cytometry. A replacement ofHVR1 with the PyCS-B epitope clearly made the adenovirus resilient toanti-adenovirus serotype 5 sera, whereas the modification of HVR5 orFiber had no effect (FIG. 55).

Next, it was determined whether HVR1 is a critical molecule for theneutralization in vivo. For this purpose, mice were infected with 1×10¹⁰v.p. wt/Empty adenovirus twice to mount sufficient pre-existinganti-adenovirus immunity (FIG. 56A) and randomized based on theiranti-adenovirus antibody titers, as determined by ELISA. The mice werethen given a single immunizing dose of capsid-modified adenovirus orunmodified adenovirus, and the level of PyCS-specific CD8+ T cellresponse was measured as described above. Only vaccination with(QGPGAP)₃-HVR1/PyCS-GFP or (QGPGAP)₃-HVR1/Fib/PyCS-GFP was able toinduce a significantly more potent CS-specific CD8+ T cell response,compared to that induced by other capsid-modified or unmodifiedadenovirus (FIG. 56B).

The level of antibody response against (QGPGAP)₃ epitope was alsomeasured, which is expressed on the capsid proteins of rAd, in miceinfected with wt/Empty Ad followed by vaccination with capsid-modifiedrAd (FIG. 57A). Only mice vaccinated with (QGPGAP)₃—HVR1/PyCS-GFP and(QGPGAP)₃—HVR1/Fib/PyCS-GFP were able to mount a significantly highertiter of anti-QGPGAP antibody than those vaccinated with wt/PyCS-GFP(FIG. 57B).

The examples described above are meant to more fully illustrate theembodiments and are not to be interpreted as limiting the scope of anyclaimed embodiment. In addition, the references cited within thedisclosure, and all references listed below are hereby incorporated byreference in their entirety as if fully set forth herein.

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What is claimed is:
 1. A recombinant adenovirus derived from arecombinant adenovirus plasmid vector, wherein the recombinantadenovirus plasmid vector comprises a nucleotide sequence encoding: aPlasmodium falciparum circumsporozoite protein gene, or antigenicportion thereof, operably linked to a heterologous promoter sequence,and one or more modified capsid and/or core protein genes, wherein animmunogenic epitope sequence of Plasmodium falciparum circumsporozoitehas been inserted into or replaces at least part of the one or morecapsid and/or core protein genes; wherein the immunogenic epitopesequence is a B cell epitope sequence selected from (NANP)₁₂, (NANP)₁₄,(NANP)₁₆, (NANP)₁₈, (NANP)₂₀, (NANP)₂₂; and wherein the core proteingene further comprises a pVII protein gene and a CD4+T cell epitopesequence that is inserted into the pVII protein gene.
 2. The adenovirusof claim 1, wherein the Plasmodium falciparum circumsporozoite proteingene further comprises a codon-optimized Plasmodium falciparumcircumsporozoite protein gene.
 3. A recombinant adenovirus derived froma recombinant adenovirus plasmid vector, wherein the recombinantadenovirus plasmid vector comprises a nucleotide sequence encoding: aPlasmodium falciparum circumsporozoite protein gene, or antigenicportion thereof, operably linked to a heterologous promoter sequence,and one or more modified capsid and/or core protein genes, wherein animmunogenic epitope sequence of Plasmodium falciparum circumsporozoitehas been inserted into or replaces at least part of the one or morecapsid and/or core protein genes; wherein the immunogenic epitopesequence is a B cell epitope sequence selected from (NANP)₁₂, (NANP)₁₄,(NANP)₁₆, (NANP)₁₈, (NANP)₂₀, (NANP)₂₂; and wherein the Plasmodiumfalciparum circumsporozoite protein gene further comprises acodon-optimized Plasmodium falciparum circumsporozoite protein geneencoded by SEQ ID NO:2.
 4. The adenovirus of claim 1, wherein the capsidprotein gene further comprises a Hexon hypervariable region (HVR)sequence.
 5. The adenovirus of claim 4, wherein the HVR sequence furthercomprises an HVR1 or HVR5 sequence and the B cell epitope sequence: a)is inserted in the HVR1 or HVR5 sequence; or b) replaces a portion ofthe HVR1 or HVR5 sequence.
 6. A recombinant adenovirus derived from arecombinant adenovirus plasmid vector, wherein the recombinantadenovirus plasmid vector comprises a nucleotide sequence encoding: aPlasmodium falciparum circumsporozoite protein gene, or antigenicportion thereof, operably linked to a heterologous promoter sequence,and one or more modified capsid and/or core protein genes encoded by anucleic acid sequence selected from the group consisting of SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, or SEQ ID NO:22, wherein an immunogenic epitope sequence ofPlasmodium falciparum circumsporozoite has been inserted into orreplaces at least part of the one or more capsid and/or core proteingenes; wherein the immunogenic epitope sequence is a B cell epitopesequence selected from (NANP)₁₂, (NANP)₁₄, (NANP)₁₆, (NANP)₁₈, (NANP)₂₀,(NANP)₂₂, wherein the capsid protein gene further comprises a Hexonhypervariable region (HVR) sequence; and wherein the HVR sequencefurther comprises an HVR1 or HVR5 sequence and the B cell epitopesequence: a) is inserted in the HVR1 or HVR5 sequence; or b) replaces aportion of the HVR1 or HVR5 sequence.
 7. The adenovirus of claim 1,wherein the one or more modified capsid and/or core protein genescomprises a capsid Fiber protein gene and the B cell epitope sequence isinserted into the Fiber protein gene.
 8. A recombinant adenovirusderived from a recombinant adenovirus plasmid vector, wherein therecombinant adenovirus plasmid vector comprises a nucleotide sequenceencoding: a Plasmodium falciparum circumsporozoite protein gene, orantigenic portion thereof, operably linked to a heterologous promotersequence, and one or more modified capsid and/or core protein genescomprising a capsid Fiber protein gene and the B cell epitope sequenceis inserted into the Fiber protein gene, wherein an immunogenic epitopesequence of Plasmodium falciparum circumsporozoite has been insertedinto or replaces at least part of the one or more capsid and/or coreprotein genes; wherein the immunogenic epitope sequence is a B cellepitope sequence selected from (NANP)₁₂, (NANP)₁₄, (NANP)₁₆, (NANP)₁₈,(NANP)₂₀, (NANP)₂₂; and wherein the modified capsid protein gene isencoded by SEQ ID NO:24 or SEQ ID NO:25.
 9. A recombinant adenovirusderived from a recombinant adenovirus plasmid vector, wherein therecombinant adenovirus plasmid vector comprises a nucleotide sequenceencoding: a Plasmodium falciparum circumsporozoite protein gene, orantigenic portion thereof, operably linked to a heterologous promotersequence, and one or more modified capsid and/or core protein genescomprising a capsid Fiber protein gene and the B cell epitope sequenceis inserted into the Fiber protein gene, wherein an immunogenic epitopesequence of Plasmodium falciparum circumsporozoite has been insertedinto or replaces at least part of the one or more capsid and/or coreprotein genes; wherein the immunogenic epitope sequence is a B cellepitope sequence selected from (NANP)₁₂, (NANP)₁₄, (NANP)₁₆, (NANP)₁₈,(NANP)₂₀, (NANP)₂₂; wherein the core protein gene further comprises apVII protein gene and a CD4+T cell epitope sequence that is insertedinto the pVII protein gene; and wherein the modified core protein geneis encoded by SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.10. The adenovirus of claim 1 or claim 9, wherein the CD4+T cell epitopesequence is a Plasmodium falciparum circumsporozoite protein gene CD4+Tcell epitope sequence.
 11. The adenovirus of claim 1 or 9, wherein theCD4+T cell epitope sequence is EYLNKIQNSLSTEWSPCSVT (SEQ ID NO:62). 12.The adenovirus of claim 1, wherein the adenovirus is produced from anyone of the recombinant adenovirus plasmid vectors selected from thegroup consisting of an HVR1-modified adenovirus vector, a Fiber-modifiedadenovirus vector, an HVR1 and Fiber-modified adenovirus vector, a Fiberand pVII-modified adenovirus vector, an HVR1 and pVII-modifiedadenovirus vector and an HVR1, Fiber and pVII-modified adenovirusvector.
 13. A recombinant adenovirus derived from a recombinantadenovirus plasmid vector, wherein the recombinant adenovirus plasmidvector comprises a nucleotide sequence encoding: a Plasmodium falciparumcircumsporozoite protein gene, or antigenic portion thereof, operablylinked to a heterologous promoter sequence, and one or more modifiedcapsid and/or core protein genes, wherein an immunogenic epitopesequence of Plasmodium falciparum circumsporozoite has been insertedinto or replaces at least part of the one or more capsid and/or coreprotein genes; and wherein the core protein gene comprises a pVIIprotein gene and a CD4+T cell epitope sequence is inserted into the pVIIprotein gene.
 14. The adenovirus of claim 13 wherein the modified coreprotein gene is encoded by SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.15. The adenovirus as in any of claims 13, wherein the CD4+T cellepitope sequence is a Plasmodium falciparum circumsporozoite proteingene CD4+T cell epitope sequence.
 16. The adenovirus of claim 13,wherein the CD4+T cell epitope sequence is EYLNKIQNSLSTEWSPCSVT (SEQ IDNO:62).